Histone H4
The abnormal oocyte (abo) gene of Drosophila is a peculiar maternal effect gene whose mutations cause a maternal-effect lethality that can be rescued by specific regions of heterochromatin during early embryogenesis. An increase in the dosage of specific regions of heterochromatin, denoted ABO, to either the mutant mother or the zygote, increases embryonic survival rates. abo encodes an evolutionary
conserved chromosomal protein that localizes exclusively to the histone gene cluster and binds to the regulatory regions of such genes. A significant increase of histone transcripts occurs in eggs of abo mutant mothers and a partial rescue of the abo maternal-effect defect takes place with
deficiencies of the histone gene cluster. On the basis of these results, it is suggested that the Abo protein functions specifically as a negative regulator
of histone transcription and a molecular model is proposed to account for the ability of heterochromatin to partially rescue the abo maternal-effect defect. This model proposes that increased doses of specific heterochromatic regions titrate out abnormally high levels of histones present in embryos from mutant abo mothers and that a balanced pool of histones is critical for normal embryogenesis in Drosophila (Berloco, 2001).
The abo gene consists of a 1,974-bp sequence containing a putative TATA box, a CAAT box, and an ORF, interrupted by a small intron, and
producing a single 1.8-kb transcript. This transcript encodes a putative 509-aa protein. The abo1 mutation is due to the insertion of
an incomplete Doc transposable element into the coding
region of the abo gene producing a larger transcript than
the wild type, whereas that abo2 mutation is caused by a
P[ry+] insertion into the 5'
promoter region and does not produce a detectable transcript (Berloco, 2001).
A computer database search (the BLASTP program) found
no known protein motifs in the conceptually translated Abo protein. However, 25.3% identity and 51.9% similarity was found to the DET1 protein, a
nuclear located negative regulator of light-mediated gene expression in Arabidopsis, whose putative homologs are present also in Oryza sativa and Lycopersicon esculentum. Intriguingly,
24% identity and 44% similarity was found to the putative human hCP43420
protein from the Celera Human Report and to a putative mouse protein. Considering the
evolutionary distance, the homology between these proteins appears
significant. They share stretches of homology across their entire
lengths and are very similar in charge, distribution of hydrophilic
residues, and overall amino acid composition. In particular, the human
and mouse proteins appear strikingly identical, with few differences in
the nucleotide sequences of their encoding genes (Berloco, 2001).
The homology with DET1 suggests that the Abo protein might also be a
transcriptional regulator and therefore might bind specific target
sequences. To test this, bacterially produced Abo protein was used as an
antigen to raise a polyclonal antibody in mice. Both the polytene chromosomes from salivary glands and
the mitotic chromosomes of neuroblasts from wild-type larvae stain for Abo protein. A strong signal exclusively localized on the 39E region on
polytene chromosomes was seen. In mitotic metaphase
chromosomes, a unique strong signal is present on the constriction on
the base of the left arm of the second chromosome.
In both cases, the signal is localized at the position of the histone gene cluster, as confirmed by sequential immunostaining with the anti-Abo antibodies and in situ hybridization of the cDm500
probe, which contains the histone cluster. These results clearly demonstrate that the regions with
exclusive binding affinity for Abo contain the histone clusters in both
the polytenes and mitotic chromosomes (Berloco, 2001).
To identify Abo-binding sites in the histone repeat unit,
the X-ChIP (formaldehyde-crosslinked-chromatin immunoprecipitation) method was applied by using polyclonal anti-Abo antibodies. 12 overlapping primer pairs were designed that amplify 400- to 500-bp fragments spanning
the whole Drosophila histone repeat unit and they were used to amplify
the DNA immunoprecipitated from chromatin of early embryos (0-4 h old)
and SL-2 cultured cells. Binding was found of Abo protein in early
embryos to the promoter regions of H2A-H2B and H3-H4. In SL-2 cells, Abo binds to an additional site in an H1 promoter fragment.
These results show clearly that Abo protein binding is restricted to
the three main regulatory regions of the repeat unit containing the
histone gene promoters (Berloco, 2001).
The functional significance of the interaction of abo with
the promoters of histone genes was addressed by a quantitation of
histone transcripts in unfertilized eggs from heterozygote abo1/abo2 and
abo1/abo+ mothers. The results show that abo mutations affect histone transcription. Much higher levels of H2A and H2B were found in eggs from mutant mothers than in eggs from their heterozygous sisters. The amount of H3 and H4 transcripts was significantly higher, whereas variations in the amount of H1 transcripts were not detectable. These results strongly suggest that abo is a negative regulator of histone genes. This possibility was further examined by testing the genetic effects of deficiencies of the entire histone gene cluster on the
abo1 maternal effect. The results clearly show that the
histone deficiencies [Df(2)DS5 and Df(2)DS6]
induce a strong suppression of the abo1
maternal-effect defect, thus giving strong support to the suggestion that Abo negatively regulates histone gene expression (Berloco, 2001).
Taken together, these studies reveal that abo is a
negative regulator of H2A, H2B, H3, and H4 expression during oogenesis.
Hence, the deleterious maternal-effect defect induced by the
abo mutations is probably due to an excess of these
histones. The regulation of histone expression has been extensively
studied in different species. The 5' flanking regions contain
cis elements that interact with transacting factors. These transacting
factors differ among species and, more surprisingly, also differ among the different classes of histone genes. It has been proposed that the
coordinate expression of the histone genes probably depends on the
interaction of a protein complex with the different transacting factors. In this context, the uniqueness of the Abo protein location on
the histone genes in different Drosophila species and its
strong evolutionarily conservation suggest that this protein probably
plays a basic role in regulating histone gene expression. However,
differential histone gene expression in early embryogenesis of several
species has been seen. In Drosophila, specific histone classes are also known to be differentially expressed. For
example, it has been shown that the maternal histone H1 transcript is
not translated in early embryogenesis and is replaced by the HMG-D
chromosomal protein. Intriguingly, the lack of any effect on H1
histone maternal transcription by the abo mutations and the
lack of binding to its promoter by Abo in early embryos suggest that
the regulation of histone H1 in both ovaries and embryos could not
involve the abo gene. However, Abo does bind to the H1
promoter in SL-2 cells (representing late embryonic tissue), suggesting
that Abo is probably involved in transcriptional regulation of histone
H1 later in embryogenesis. Moreover, the differential enhancement of
transcripts found in eggs from abo mutant mothers
suggests that Abo could be more important for H2A and H2B repression
than H3 and H4 repression during oogenesis (Berloco, 2001).
The data suggest a simple direct model for explaining an
intriguing aspect of this gene, namely its interaction with the
specific heterochromatic regions termed ABO elements.
According to the model, homozygous abo mothers produce eggs
with disproportionately high levels of H2A, H2B, H3, and H4 histones,
which affect egg viability. Increasing doses of the ABO
regions may titrate out these histones, reducing their negative effect.
It is predicted that the abo and ABO-counteracting
effects are produced by modulations in chromatin structure. Histones
could be involved in such effects, as suggested by growing evidence
showing that modified histones have differential chromosomal
distributions, and hence they could play a role in the formation of
heterochromatic domains. In fact H4 histone acetylated at lysine 4 and H3 histone methylated at lysine 9 are both present along the mitotic heterochromatin of Drosophila, with patterns of distribution indicating preferential binding for some regions (Berloco, 2001).
In conclusion, the characterization of abo opens the
possibility of using this gene as an entry point to dissect the regulatory machinery of histone expression by looking at Abo-interacting molecules. Moreover, it could be a paradigm for experimental approaches to study the biological role of the heterochromatin. In D. melanogaster, other maternal-effect mutations closely linked to abo have been isolated.
Preliminary experiments provide evidence that these abo-like
mutations produce defects that can be compensated by discrete
heterochromatic elements similar to ABO. It is possible that these other genes, like abo, may also encode transregulators of histone genes or other essential genes encoding chromosomal proteins (Berloco, 2001).
Nuclear extracts from Drosophila Kc cells were used to characterize 3' end processing of Drosophila histone pre-mRNAs. Drosophila Stem-loop binding protein (SLBP) plays a critical role in recruiting the U7 snRNP (Dominski, 2003) to the pre-mRNA and is essential for processing all five Drosophila histone pre-mRNAs. The Drosophila processing machinery strongly prefers cleavage after a fourth nucleotide following the stem-loop and favors an adenosine over pyrimidines in this position. Increasing the distance between the stem-loop and the histone downstream element (HDE) does not result in a corresponding shift of the cleavage site, suggesting that in Drosophila processing the U7 snRNP does not function as a molecular ruler. Instead, SLBP directs the cleavage site close to the stem-loop. The upstream cleavage product generated in Drosophila nuclear extracts contains a 3' OH, and the downstream cleavage product is degraded by a nuclease dependent on the U7 snRNP, suggesting that the cleavage factor has been conserved between Drosophila and mammalian processing. A 2'O-methyl oligonucleotide complementary to the first 17 nt of the Drosophila U7 snRNA was not able to deplete the U7 snRNP from Drosophila nuclear extracts, suggesting that the 5' end of the Drosophila U7 snRNA is inaccessible. This oligonucleotide selectively inhibited processing of only two Drosophila pre-mRNAs and had no effect on processing of the other three pre-mRNAs. Together, these studies demonstrate that although Drosophila and mammalian histone pre-mRNA processing share common features, there are also significant differences, likely reflecting divergence in the mechanism of 3' end processing between vertebrates and invertebrates (Dominski, 2005).
Metazoan replication-dependent histone pre-mRNAs do not contain introns, and the only processing reaction necessary to generate mature histone mRNAs is a single endonucleolytic cleavage of the mRNA precursors (pre-mRNAs) to form the 3' end. Studies on 3' end processing were initially carried out in Xenopus oocytes using synthetic pre-mRNAs and sea urchin histone genes and later were facilitated by the development of an in vitro system based on nuclear extracts from mammalian cells. Replication-dependent histone pre-mRNAs contain two cis elements required for 3' end processing: a highly conserved stem-loop structure consisting of a 6-bp stem and a 4-nt loop and a less conserved histone downstream element (HDE) located ~15 nt 3' of the stem-loop. Mammalian histone pre-mRNAs are cleaved between the two elements, 5 nucleotides downstream of the stem-loop. The stem-loop is recognized by the stem-loop binding protein (SLBP), also referred to as the hairpin binding protein (HBP). The HDE interacts with the U7 snRNP, which contains an ~60-nt U7 snRNA, and this interaction is primarily mediated by base-pairing between the HDE and the 5' end of U7 snRNA. In vitro studies in mammalian nuclear extracts suggest that SLBP stabilizes binding of the U7 snRNP to the pre-mRNA and is essential in processing of only those pre-mRNAs that do not form sufficiently stable duplexes with the U7 snRNA. This role of SLBP in mammalian processing is most likely mediated by ZFP100, a 100-kDa zinc finger protein associated with the U7 snRNP and interacting with the SLBP/stem-loop complex. In addition to bridging the two factors bound to their respective sequence elements, ZFP100 may also play other roles in 3' end processing, possibly including the recruitment of the cleavage factor (Dominski, 2005).
Purification of the U7 snRNP from mammalian cells resulted in identification of two novel Sm-like proteins: Lsm10 and Lsm11, which replace the D1 and D2 Sm proteins present in the spliceosomal snRNPs. Lsm11 interacts in vitro with ZFP100 and plays a key role in recognizing the unique sequence of the Sm binding site in U7 snRNA. Orthologs of Lsm10 and Lsm11 are also found in the Drosophila U7 snRNP, demonstrating that the unique structure of the U7 snRNP in vertebrates and invertebrates is conserved. A counterpart of ZFP100 has not been yet identified in the Drosophila genome, suggesting that ZFP100 is either weakly conserved between vertebrates and invertebrates or processing of histone pre-mRNAs in Drosophila does not require this protein (Dominski, 2005).
Nuclear extracts from Drosophila S-2 and Kc cultured cells and embryos are capable of 3' end processing of presynthesized Drosophila histone pre-mRNAs. Nuclear extracts from Kc cells are also capable of cotranscriptional processing of histone pre-mRNAs. Unlike the auxiliary role played by SLBP in mammalian in vitro processing, Drosophila SLBP is indispensable for processing of all Drosophila histone pre-mRNAs. This observation suggests that Drosophila SLBP plays a much more important role in recruiting the U7 snRNP to the pre-mRNA than it does in the mammalian processing. This study uses an in vitro system based on Drosophila nuclear extracts to characterize 3' end processing of Drosophila histone pre-mRNAs and to define differences and similarities in processing between this model invertebrate processing system and processing in mammalian nuclear extracts (Dominski, 2005).
These studies demonstrate that although Drosophila and mammalian histone pre-mRNA processing occur with similar chemistry and both require SLBP and the U7 snRNP, the two mechanisms differ significantly in the relative importance of these trans-acting factors and in the specification of the cleavage site (Dominski, 2005).
Drosophila nuclear extracts cleave histone pre-mRNAs after the fourth nucleotide following the stem-loop and prefer an adenosine preceding the cleavage site. Consistent with this, all natural Drosophila histone pre-mRNAs contain an adenosine in this position. If the fourth nucleotide is changed to a pyrimidine, cleavage is also efficient after an adenosine at the third position but not after an adenosine located 5 nt downstream of the stem-loop, i.e., at the site exclusively utilized during mammalian processing. Sea urchin histone mRNAs, the only other invertebrate histone mRNAs with the characterized 3' ends, terminate with an ACCA consensus sequence. Thus, cleavage after the fourth nucleotide following the stem-loop may be a general feature of 3' end processing of invertebrate histone pre-mRNAs. Both Drosophila and mammalian processing machineries are similar in their extreme resistance to EDTA, generation of a 3' hydroxyl group at the end of the upstream cleavage product, and degradation of the downstream cleavage product by a U7 snRNP dependent activity. These results suggest that both processing machineries utilize the same or a highly related cleavage factor in 3' end processing of histone pre-mRNAs (Dominski, 2005).
In mammalian processing, the site of cleavage is determined by the position of the HDE, and moving the HDE, and, hence, the U7 snRNP, away from the stem-loop by as few as 4 nt results in a corresponding shift of the cleavage site. This observation led to the hypothesis that U7 snRNP recruits the cleavage factor to the pre-mRNA and acts as a molecular ruler to specify the cleavage site. SLBP bound to the stem-loop facilitates binding of the U7 snRNP to the HDE but does not play a direct role in recruitment of the cleavage factor. Consistent with this model, removal of SLBP, or using a substrate that cannot bind SLBP, reduces processing activity but does not abolish it (Dominski, 2005).
In contrast to mammalian processing, processing of Drosophila histone pre-mRNA is absolutely dependent on SLBP. In addition, increasing the distance between the stem-loop and the HDE by 4 or 8 nt in Drosophila histone pre-mRNA moved the cleavage site only 1 nt upstream from its normal position and did not abolish processing at the normal site. Larger insertions between the stem-loop and the HDE resulted in low efficiency cleavage further away from the stem-loop, but cleavage at these sites was still dependent on SLBP. This is in direct contrast to mammalian histone processing, where cleavage at the distant sites is independent of SLBP. Thus, in Drosophila processing the U7 snRNP does not function as a molecular ruler, but instead SLBP plays the critical role in specifying the cleavage site (Dominski, 2005).
To explain the observed differences between processing in Drosophila and mammalian nuclear extracts, it is proposed that within the Drosophila processing complex SLBP tightly interacts with the U7 snRNP, and this interaction is essential for bringing the U7 snRNP to the pre-mRNA. The two factors remain associated even if their respective binding sites are separated by a larger distance, likely by looping out the inserted nucleotides. The mutant pre-mRNAs are preferentially cleaved close to the stem-loop, reflecting the critical role of SLBP in forming the processing complex, although the precise position of the cleavage site and efficiency of processing depends on the size of the insert. In mammalian processing, the region between the stem-loop and the HDE is either rigidified, thus precluding looping out the inserted nucleotides, as previously suggested, or the interaction between SLBP and the U7 snRNP is relatively weak and disrupted by larger insertions, so binding of the U7 snRNP to the pre-mRNA depends solely on the base-pairing interaction. It is likely that in Drosophila processing the cleavage factor is recruited to histone pre-mRNA by interaction with both the U7 snRNP and SLBP, and neither factor is competent to carry out this function individually (Dominski, 2005).
In mammalian nuclear extracts, processing of histone pre-mRNAs is efficiently inhibited by relatively short 2'O-methyl oligonucleotides complementary to the 5' end of the mammalian U7 snRNA. These oligonucleotides, including a 10-mer, are also very efficient in depleting the U7 snRNP from nuclear extracts and were successfully used to affinity purify U7 snRNP from mammalian cells, demonstrating that the 5' end of the mammalian U7 snRNA is readily accessible. In contrast, two relatively long oligonucleotides, alphaDa, complementary to the first 17 nt of the Drosophila U7 snRNA, and alphaDb, complementary to nt 4-23, were not effective in depleting the U7 snRNP from Drosophila nuclear extracts. These results suggest that the 5' end of U7 snRNA is not accessible in the Drosophila U7 snRNP (Dominski, 2005).
Surprisingly, the alphaDa 2'O-methyl oligonucleotide abolished processing of the dH3* and dH1* pre-mRNAs (hybrid pre-mRNAs consisting of the stem-loop and cleavage site from the mouse H2a-614 pre-mRNA) but did not significantly affect processing of the other three Drosophila histone pre-mRNAs. Three additional oligonucleotides complementary to the regions of the U7 snRNP located closer to the Sm binding site effectively blocked processing of all five histone pre-mRNAs. It is not understood why processing of only two Drosophila pre-mRNAs is affected by the alphaDa oligonucleotide and which features of the HDEs make processing of the Drosophila pre-mRNAs either sensitive or resistant to this oligonucleotide. Selective inhibition of processing by the alphaDa oligonucleotide depending on the type of pre-mRNA used in the reaction suggests that blocking of the U7 snRNA must occur during processing. One possibility is that the U7 snRNP is initially recruited to the pre-mRNA solely by SLBP bound to the pre-mRNA, and later this interaction is followed by formation of a duplex between the HDE and the U7 snRNA, as a result of unmasking of the 5' end of U7 snRNA. The alphaDa oligonucleotide might block binding of the U7 snRNA to the HDE in the hybrid dH1* and dH3* pre-mRNAs, but not in the other pre-mRNAs, during this later step, while the other oligonucleotides block binding to all the HDEs (Dominski, 2005).
Overall, thes studies indicate that the structure of the 5' end of the Drosophila U7 snRNA and the mechanism of its initial interactions with the HDE differ significantly from the recognition of the HDE in processing of mammalian histone pre-mRNAs (Dominski, 2005).
In vitro processing of all five Drosophila histone pre-mRNAs is absolutely dependent on SLBP. This study has demonstrated that SLBP is essential for recruitment of the U7 snRNP to the pre-mRNA. The necessity of SLBP for recruitment of the U7snRNP to the Drosophila pre-mRNAs suggests that either Drosophila HDEs are unable to form a strong duplex with the U7 snRNA or that the interaction of the U7 snRNP with the SLBP/pre-mRNA complex is necessary to promote base-pairing by making the 5' end of U7 snRNA accessible (Dominski, 2005).
Both the 5' end of the Drosophila U7 snRNA and Drosophila HDEs are AU rich, allowing a number of possible base-pair schemes for making a duplex between the two RNAs. It is hypothesized that the most likely alignment used during processing is the one that allows formation of the largest number of base pairs between the purine core of the HDE and the CUCUUU sequence in the U7 snRNA and not necessarily the alignment that allows formation of the overall most stable duplex. The CUCUUU sequence is highly conserved among all known U7 snRNAs and is involved in recognition of the purine core in sea urchin and mammalian histone pre-mRNAs. A 3-nt mutation within the purine core of the hybrid dH3* pre-mRNA abolishes processing, whereas a 6-nt mutation within the AU-rich region immediately downstream of the purine core only partially inhibits processing. These results support the interpretation that base-pairing between the U7 snRNA and the purine core is critical, whereas formation of additional base in other regions increases the efficiency of Drosophila processing. It is also possible that the base-pairing interaction is limited to the purine core and the CUCUUU sequence in the U7 snRNA, whereas the AU-rich sequences in the U7 snRNA and the HDE are brought together by protein-protein interactions (Dominski, 2005).
This study demonstrated that the HDE of the hybrid dH3* pre-mRNA can abolish processing of the full-length substrate, presumably by sequestering the U7 snRNP, only when present at very high concentrations. Interestingly, this weak interaction of Drosophila HDEs with the U7 snRNP is sufficient to recruit a 5'-3' exonuclease that specifically degrades the downstream cleavage product in a U7 dependent manner. Thus, the endonucleolytic cleavage must require much stronger binding of the U7 snRNP to the pre-mRNA, while degradation of the DCP by an exonuclease may require only loose association of the HDE with the U7 snRNP (Dominski, 2005).
The most notable difference between histone pre-mRNA processing in Drosophila and mammalian nuclear extracts is the absolute dependence of Drosophila processing on SLBP and the role of SLBP in specifying the cleavage site close to the stem-loop. The Drosophila U7 snRNP does not function as a molecular ruler in processing and this feature most likely reflects a critical role of SLBP in recruiting the cleavage factor as well as the U7 snRNP, to histone pre-mRNA. These data suggest that SLBP and the U7 snRNP may form a tight complex on the histone pre-mRNA, and this complex remains stable even in the presence of large insertions between the stem-loop and the HDE (Dominski, 2005).
The similarities in the chemistry of the cleavage reaction, including preference for an adenosine preceding the cleavage site and generation of the 3'OH group in the presence of EDTA, as well as degradation of the downstream cleavage product by a U7-dependent 5'-3' exonuclease suggest that the cleavage factor has been conserved between Drosophila and mammalian processing. It will be of interest to determine whether there are factors unique to only one of these two types of organisms emphasizing long evolutionary distance and the divergence between vertebrates and invertebrates (Dominski, 2005).
Metazoan replication-dependent histone mRNAs are not polyadenylated, and instead terminate in a conserved stem–loop structure generated by an endonucleolytic cleavage involving the U7 snRNP, which interacts with histone pre-mRNAs through base-pairing between U7 snRNA and a purine-rich sequence in the pre-mRNA located downstream of the cleavage site. Null mutations of the single Drosophila U7 gene were generated and U7 snRNA was demonstrated to be required in vivo for processing all replication-associated histone pre-mRNAs. Mutation of U7 results in the production of poly A+ histone mRNA in both proliferating and endocycling cells because of read-through to cryptic polyadenylation sites found downstream of each Drosophila histone gene. A similar molecular phenotype also results from mutation of Slbp, which encodes the protein that binds the histone mRNA 3' stem–loop. U7 null mutants develop into sterile males and females, and these females display defects during oogenesis similar to germ line clones of Slbp null cells. In contrast to U7 mutants, Slbp null mutations cause lethality. This may reflect a later onset of the histone pre-mRNA processing defect in U7 mutants compared to Slbp mutants, due to maternal stores of U7 snRNA. A double mutant combination of a viable, hypomorphic Slbp allele and a viable U7 null allele is lethal, and these double mutants express polyadenylated histone mRNAs earlier in development than either single mutant. These data suggest that SLBP and U7 snRNP cooperate in the production of histone mRNA in vivo, and that disruption of histone pre-mRNA processing is detrimental to development (Godfrey, 2006).
Chromosome duplication during the cell cycle requires the production of histones during S phase to package newly replicated DNA into chromatin. Bulk histone production during S phase is achieved through the biosynthesis of replication-dependent histone mRNAs, which are cell-cycle regulated and accumulate only in S phase. In animal cells these histone mRNAs are unique: The 3' end terminates in a conserved 26-nt sequence that forms a stem–loop rather than in a poly A+ tail. Since histone genes lack introns, the only processing step required for mature histone mRNA production is endonucleolytic cleavage of the pre-mRNA to form the 3' end of the mRNA. Much of the cell-cycle regulation of histone mRNAs is post-transcriptional and is mediated by the 3' end of the mRNA. Thus, a complete understanding of cell-cycle-regulated histone mRNA production requires a full understanding of the factors required for histone pre-mRNA processing (Godfrey, 2006).
The processing of histone pre-mRNAs requires two cis elements and a number of trans-acting factors. The cis elements are the stem–loop at the 3' end of histone mRNA and a purine-rich region downstream of the cleavage site, termed the histone downstream element (HDE). A protein called stem–loop binding protein (SLBP) or hairpin binding protein (HBP) specifically binds the 3' end of histone mRNA. SLBP is required for histone pre-mRNA processing in vivo and accompanies the mRNA to the cytoplasm, where it promotes the translation of the histone mRNA. The HDE binds U7 snRNP by base-pairing with the 5' end of U7 snRNA. In mammals, SLBP, the U7 snRNP, and a U7 snRNP-associated zinc finger protein called ZFP100 cooperate to recruit an endonuclease complex that cleaves the pre-mRNA. Recent evidence indicates that CPSF73, a component of the complex that mediates AAUAAA-directed cleavage prior to polyadenylation, is the likely endonuclease. This revealed some unexpected overlap in the machinery carrying out histone pre-mRNA processing and canonical polyadenylation (Godfrey, 2006).
The U7 snRNA is a small RNA (55–70 nt) that, like the spliceosomal snRNAs, contains both a trimethyl guanosine cap and an Sm binding site, which is essential for its function. The Sm site in these snRNAs stably binds a complex of seven related proteins of the LSm/Sm family to form the core snRNP particle. Proteins of the LSm/Sm family share a common tertiary structure called the Sm fold that assembles into hexameric or heptameric rings capable of binding single-stranded RNA. The U snRNPs contain a heptameric Sm ring, with each of the seven individual subunits making a specific contact with a residue in the Sm binding site of the snRNA. The heptameric Sm ring of spliceosomal snRNPs contains the proteins SmB/B', SmD1, SmD2, SmD3, SmE, SmF, and SmG. In contrast, the U7 snRNP contains five of these Sm proteins (B/B1, D3, E, F, G) and two novel Sm proteins called LSm10 and LSm11 that replace SmD1 and SmD2 of the spliceosomal snRNPs. The Sm site found in U7 snRNAs is distinct from the Sm site in spliceosomal snRNAs and is responsible for incorporation of LSm10 and LSm11 into the U7 snRNP. In addition to the Sm fold that participates in ring formation, LSm11 contains an NH2 terminal extension that makes contacts with ZFP100 and possibly other components of the histone pre-mRNA processing machinery (Godfrey, 2006).
The role of U7 snRNP in histone pre-mRNA processing has been examined primarily in nuclear extract systems that support the processing of synthetic histone pre-mRNAs, and by monitoring the processing of histone pre-mRNAs injected into Xenopus ooctyes. Complementary mutations in U7 snRNA and the HDE provided early evidence that base-pairing between the 5' end of U7 and the HDE was an important part of U7 snRNP function. Furthermore, blocking the 5' end of the U7 snRNA with a complementary oligonucleotide specifically inhibits processing of synthetic histone pre-mRNAs in nuclear extracts. However, the contribution of U7 snRNA to endogenous histone mRNA biosynthesis and whether this contribution is important for animal development have not been examined. To explore these issues, U7 snRNA mutations in Drosophila were generated and characterized (Godfrey, 2006).
Drosophila SLBP, U7 snRNA, and U7 snRNP specific proteins Lsm10 and Lsm11, have all been identified, and steps have been taken to characterize them genetically. Mutations in the Drosophila Slbp gene block normal histone pre-mRNA processing during embryonic development and result in production of polyadenylated histone mRNAs as a consequence of read-through past the normal processing site. This occurs because each of the five Drosophila histone genes contains cryptic polyadenylation sites downstream of the HDE that are utilized in the absence of SLBP. Null mutations of Slbp cause lethality during larval and pupal stages, presumably because of the histone processing defects, although the precise cause of lethality is not known. Slbp mutant cells are capable of replicating chromatin, likely because the inappropriate polyadenylated mRNAs are translated. A hypomorphic Slbp mutant allele that produces reduced amounts of SLBP protein results in the production of both normal and poly A+ histone mRNAs during embryogenesis, but does not cause lethality. However, these viable mutant females lay eggs that contain reduced amounts of histone mRNA and protein and do not develop. Thus, SLBP is required during both zygotic development and oogenesis (Godfrey, 2006).
This study compared mutations in the U7 snRNA gene, and the resulting phenotypes were compared with those caused by mutation of Slbp. The results indicate that U7 snRNA is required for normal histone mRNA biosynthesis during Drosophila development and that, like Slbp mutations, loss of U7 snRNA results in the production of polyadenylated histone mRNAs. However, unlike Slbp null mutants, U7 null mutants are viable, but both males and females are sterile. This difference in terminal phenotype is most likely because the maternal supply of U7 snRNA delays the onset of the histone processing defect in U7 mutants relative to Slbp mutants, which do not have a significant maternal supply of SLBP protein. Both U7 and SLBP are required for normal histone mRNA biosynthesis in the female germ line, and mutation of either gene disrupts oogenesis. These data indicate that loss of SLBP and U7 cause similar molecular phenotypes in Drosophila and suggest that early expression of this molecular phenotype prevents normal development (Godfrey, 2006).
During periods of active DNA replication and chromatin assembly, newly synthesized histone H4 is
deposited in a diacetylated form. In Tetrahymena, a specific pair of residues, lysines 4 and 11, has
been shown to undergo this modification in vivo. Presumably, this reaction is
catalyzed, at least in part, by histone acetyltransferases (HAT) of the B type, cytoplasmic enzymes
displaying strong preference for free, non-chromatin-bound H4. To investigate which lysine acetylation sites are preferred in the H4 of other organisms, a cytoplasmic HAT B activity was prepared
from Drosophila embryos and used to acetylate H4 from several species. When either H4 or synthetic
NH2-terminal peptides from Tetrahymena was used, acetate was preferentially incorporated at lysine 11 with little, if any,
incorporation at other conserved, acetylatable lysines. Drosophila H4 acetate incorporation occurred preferentially on lysine 12, the residue analogous to lysine 11
in Tetrahymena. These data show remarkable preference for lysine 11/12 by the Drosophila HAT B
activity in vitro and provide support for the assertion that this activity functions to acetylate new H4,
at least in part, for deposition and chromatin assembly in vivo (Sobel, 1994).
Newly synthesized histone H4 is deposited in a diacetylated isoform in a wide variety of organisms.
In Tetrahymena a specific pair of residues, lysines 4 and 11, have been shown to undergo this
modification in vivo. The analogous residues, lysines 5 and 12 are
acetylated in Drosophila and HeLa H4. These data strongly suggest that deposition-related
acetylation sites in H4 have been highly, perhaps absolutely, conserved. In Tetrahymena and
Drosophila newly synthesized histone H3 is also deposited in several modified forms. For H3, like H4, a specific, but distinct, subset of lysines is
acetylated in these organisms. In Tetrahymena, lysines 9 and 14 are highly preferred sites of
acetylation in new H3 while in Drosophila, lysines 14 and 23 are strongly preferred. No evidence has
been obtained for acetylation of newly synthesized H3 in HeLa cells. Thus, although the pattern and
sites of deposition-related acetylation appear to be highly conserved in H4, the same does not
appear to be the case for histone H3 (Sobel, 1995).
Carnitine is a well-known naturally occurring compound, very similar to butyrate, with an essential role in
intermediary metabolism mainly at the mitochondrial level. Since butyrate inhibits the enzyme histone
deacetylase and is capable of suppressing position-effect variegation in Drosophila, carnitine was tested for a similar function in the nucleus. Carnitine derivatives are capable of suppressing the position-effect
variegation, albeit with different efficiencies. The carnitine derivatives interact lethally with Su-var(2-)1(01), a
mutation that induces hyperacetylation of histones, while hyperacetylated histones accumulate in both the
nuclei of HeLa cells and Drosophila polytene chromosomes treated with the same compounds. These results
strongly suggest that the carnitine derivatives suppress position-effect variegation by a mechanism similar to
that of butyrate. It is suggested that carnitines may have a functional role in the nucleus, probably at the
chromatin level (Fanti, 1994).
The Drosophila nucleosome remodeling factor NURF utilizes the energy of ATP hydrolysis to perturb the structure of nucleosomes and facilitate binding of
transcription factors. The ATPase activity of purified NURF is stimulated significantly more by nucleosomes than by naked DNA or histones alone, suggesting that NURF is able to recognize specific features of the nucleosome. The interaction between NURF and nucleosomes is impaired by proteolytic
removal of the N-terminal histone tails and by chemical cross-linking of nucleosomal histones. The ATPase activity of NURF is also competitively inhibited by each of the four Drosophila histone tails expressed as GST fusion proteins. A similar inhibition is observed for a histone H4 tail substituted with glutamine at four conserved, acetylatable lysines. These findings indicate a novel role for the flexible histone tails in chromatin remodeling by NURF, and this role may, in part, be independent of histone acetylation (Georgel, 1997).
What are the structural determinants of nucleosomes that are important for the activity of NURF? Based on the loss of the nucleosome-stimulated ATPase activity of
NURF and the diminution of the DNase I footprint when the histone tails are removed by limited proteolysis, it is suggested that the flexible tails of the Drosophila core histones are critical elements for interaction with NURF. This conclusion is strengthened by the inhibition of NURF ATPase activity by GST-histone fusions. The
effects of cross-linking the core histones in nucleosomes are also consistent with a contribution from the histone tails, although contributions from the globular domains of the nucleosome core histones cannot be excluded by this technique. Finally, a minor role for nucleosomal DNA is indicated by the modest inhibitory effects of DNA on the nucleosome-stimulated ATPase activity of NURF. These several determinants, individually insufficient for stimulating the ATPase activity of NURF, may be required in a combinatorial manner for achieving ATP-dependent perturbation of nucleosome structure. It will be of interest to relate the recognition of these determinants to one or more subunits of the NURF complex, and to analyze how this recognition is transduced to nucleosomal reorganization coupled with the utilization of chemical energy. Although a discrete supercomplex of NURF and a nucleosome has not been detected by native gel electrophoresis, it will also be important, when sufficient amounts of NURF become available for systematic studies, to define the interactions between NURF and nucleosomes quantitatively by biophysical methods, and to determine the histone composition of the remodeled nucleosome (Georgel, 1997).
The requirement for the Drosophila histone tails in nucleosomal interactions with NURF and the lack of strong binding specificity for structured DNA, a property
of the SWI/SNF complex, provides further evidence for separate modes of action for the NURF and SWI/SNF chromatin remodeling complexes, which share related ATPase subunits ISWI and SWI2/SNF2, and the ability to alter chromatin structure in vitro in an ATP-dependent manner. Genetic studies have shown that the histone H2A/H2B tails and the histone H3/H4 tails are essential for viability in yeast. For histones H3 and H4, the tails are also important for repression of basal transcription, for telomeric and silent mating locus repression and for activation and repression of some genes. The H3 and H4 tails have been shown to bind in vitro with the yeast silencing information regulators SIR3 and SIR4, providing direct evidence that these extended regions may form specific binding sites for protein regulators of nucleosome structure and function. Tup1, a repressor of transcription of yeast a-cell specific genes, has also been demonstrated to interact directly with the tails of histones H3 and H4. Together with the present findings, these results suggest that the flexible tails of the histone octamer serve as
common sites of interaction with several distinct nuclear protein complexes that affect nucleosome stability in a positive or negative manner (Georgel, 1997).
Other biochemical studies have demonstrated that the basic histone tails partially restrict binding of transcription factors to nucleosomal DNA. This restricted accessibility of nucleosomal DNA imposed by the histone tails can be alleviated upon neutralization of charged lysines by acetylation. However, as
indicated by the ability of the GST-yH4 (Q5,8,12,16) mutant protein to retain competitive inhibition of the nucleosome-stimulated ATPase activity of NURF, the four acetylatable lysines of histone H4 in yeast do not seem to be of crucial importance for interaction with NURF, as measured by the ATPase assay. These lysine
positions are strictly conserved in the Drosophila histone H4 tail and also undergo acetylation. Hence, the remaining conserved amino acid residues of the Drosophila histone H4 tail are likely to be involved in the interaction with NURF,
and this interaction, at least for histone H4, could be independent of the state of lysine acetylation. It should be noted that these results do not exclude an interaction between NURF and other lysine residues of the histone tails that are not subject to acetylation. Nonetheless, the observed ability of hyperacetylated nucleosomes to stimulate the ATPase activity of NURF as well as normal nucleosomes, in the case of both HeLa cell and Drosophila histones, is consistent with the possibility that NURF may act independently of the histone acetylation pathway of nucleosome destabilization. It will be of interest to elucidate, by site-directed mutagenesis, the precise nature of the interaction between NURF and the histone tails, to understand the mechanism by which this interaction leads to nucleosomal reorganization and to define the parallel or sequential nature of the pathways of nucleosome reorganization by chromatin remodeling and histone modifying activities (Georgel, 1997).
A number of activators are known to increase transcription by RNA polymerase (pol) II through protein acetylation. While the physiological substrates for these acetylases are poorly defined, possible targets include general transcription factors, activator proteins and histones. Using a cell-free system to reconstitute chromatin with increased histone acetylation levels, a direct test was performed for a causal role of histone acetylation in transcription by RNA pol II. Chromatin, containing either control or acetylated histones, was reconstituted to comparable nucleosome densities and characterized by electron microscopy after psoralen cross-linking, as well as by in vitro transcription. Chromatin was reconstituted using histones from either TSA-treated CV-1 cells, which accumulates hyperacetylated histone isoforms, or from untreated cells, in which the histones are primarily non-acetylated onto a 7.75 kb plasmid containing an hsp26 minigene. This process involves the prior depletion of the endogenous histones present in the chromatin assembly extract such that chromatin is assembled quantitatively from the input, exogenous histones. The chromatin assembly reaction generates complex chromatin containing many non-histone proteins and enzymatic activities. While H1-containing control chromatin severely represses transcription of a model hsp26 gene, highly acetylated chromatin is significantly less repressive. Acetylation of histones, and particularly of histone H4, affects transcription at the level of initiation (Nightingale, 1998).
The Drosophila Polycomb and trithorax group proteins act through chromosomal elements such as Fab-7 to maintain repressed or active gene expression,
respectively. A Fab-7 element is switched from a silenced to a mitotically heritable active state by an embryonic pulse of transcription. Here, histone H4
hyperacetylation has been found to be associated with Fab-7 after activation, suggesting that H4 hyperacetylation may be a heritable epigenetic tag of the
activated element. Activated Fab-7 enables transcription of a gene even after withdrawal of the primary transcription factor. This feature may allow epigenetic
maintenance of active states of developmental genes after decay of their early embryonic regulators (Cavalli, 1999).
Fab-7-dependent chromosomal memory of silent or open
chromatin states occurs in transgenic
Drosophila lines such as FLW-1 and FLFW-1.
These lines carry a heat shock-inducible GAL4 driver (hsp70-GAL4)
regulating a GAL4-dependent lacZ reporter (UAS-lacZ) flanked
by Fab-7 and the mini-white gene. Silencing
imposed by Fab-7 on the flanking reporter genes is dependent on the components of the PcG, since heterozygous mutant PcG
genes show a relief of white gene repression. Conversely, white gene
activity requires the trxG because heterozygous mutations in the different
members tested result in a down-regulation of expression. A
GAL4 pulse during embryogenesis can impose a mitotically stable
reprogramming of the Fab-7 cellular
memory module (CMM) from a silenced to an open
chromatin state. The maintenance of the
activated Fab-7 state is dependent on trithorax (trx) but not on Polycomb (Pc). In a heterozygous Pc-
background, Fab-7 can be switched by a GAL4 pulse and be
stably maintained, resulting in strong white expression. In contrast, a trx-
mutation completely abolishes the mitotic transmission (Cavalli, 1999).
To
assess whether the epigenetically activated Fab-7 state
correlates with a permanent loss of PcG proteins from the chromatin
template, a strong GAL4 induction pulse was administered during
embryogenesis in the FLFW-1 line. Polytene chromosomes of third instar
larvae were immunostained with antibodies directed against PcG
proteins. Surprisingly, all of the PcG proteins tested, Polycomb (Pc) and
Posterior sex combs (Psc),
Polyhomeotic (Ph), and Polycomb-like (Pcl), are still strongly bound to the Fab-7
transgene irrespective of the epigenetic state. Thus, an epigenetically
activated state can be stably propagated in the presence of the protein
components of the PcG. These data support previous observations that have
demonstrated binding of Pc at cytological sites containing potentially
active genes in polytene chromosomes and binding of Ph and Psc proteins at an actively transcribed gene in
Drosophila Schneider cells. It has been
reported that certain PcG genes may function as activators in specific
tissues and at specific developmental times by genetic analyses. Although a
role for Pc protein in the maintenance of the activated state of
Fab-7 is not observed, it may be possible that other PcG
proteins are involved in this process (Cavalli, 1999).
If it is not the removal of PcG repressors on the template, what is the
epigenetic tag that marks the activated Fab-7 state? A
single embryonic GAL4 pulse was administered to FLFW-1 embryos, and
histone acetylation of the Fab-7 transgene as a possible
mark was analyzed by immunostaining polytene chromosomes of third instar larvae with specific antibodies against the tetra-acetylated form of H4 and H3 histones. Hyperacetylation of histone
H4 is detected at the Fab-7 transgene location in larvae
derived from activated embryos, but not from control embryos raised at
18°C. In contrast to H4, no hyperacetylation of
histone H3 could be detected in activated FLFW-1 individuals (Cavalli, 1999).
Patterning transcription factors, like the products of many
segmentation genes, act only shortly on their downstream genes during
early Drosophila development, whereas the PcG/trxG memory system subsequently maintains the embryonically programmed patterns. For this reason, a test was performed to see whether embryonically activated Fab-7 can maintain expression of the reporter gene
lacZ in the absence of the primary transcription factor GAL4. The leakiness of the hsp70 promoter prevents the
complete disappearance of GAL4 protein during single fly development.
To overcome this problem, use was made of the fact that activated
Fab-7 can be efficiently propagated through meiosis in the
line FLW-1. This allows the crossing out of the
GAL4 driver to test lacZ expression in the complete absence
of GAL4 in subsequent generations. Upon crossing out
GAL4 in activated flies, 20% to 25% of the GAL4-less embryos show
substantial levels of homogeneous beta-galactosidase
expression in all embryonic cells in two consecutive generations. This percentage correlates
well with the fraction of adults showing meiotically stable
white derepression. Unfortunately,
it is not possible to also test for the meiotic inheritance of H4
hyperacetylated states because of a staining pattern with endogenous
bands at the insertion site of the transgene in the FLW-1 line.
However, the functional analysis demonstrates that epigenetic
inheritance of an active Fab-7 chromatin state results in
transcriptional activity of the UAS-lacZ reporter even in the absence
of the GAL4 transactivator (Cavalli, 1999).
A weak expression of lacZ in the absence of GAL4 may arise
from a heritable loss of PcG-mediated repression, thereby neutralizing the silencing ability of Fab-7 and consequently reflecting
the ground state of a nonrepressed chromatin template. If this were the
case, it might be expected that in flies carrying an UAS-lacZ construct without Fab-7 (pU/l5), a similar weak homogeneous beta-Gal staining
pattern would be observed in all embryos in the absence of GAL4. To
test this point, beta-Gal staining was examined in two independent lines
carrying the pU/l5 construct but no GAL4 driver. In both cases, most of the embryos were not stained or were stained in a weakly variegated fashion in random cells. This strongly suggests that meiotic inheritance of Fab-7 CMM-activated states does
not simply reflect lifting of PcG-mediated silencing but rather the inheritance of an active chromatin state, which is competent for transcriptional activation (Cavalli, 1999).
These findings show in a functional manner that trxG protein complexes recruited at a CMM relieve the
requirement for the activating factor for transcriptional maintenance.
Hyperacetylation of histone H4 has been identified as an epigenetic mark for
the activated Fab-7 state. Unlike the short-lived H4
hyperacetylation induced by transient gene activation at late developmental stages, the mark set at embryonic stages is mitotically stable and inheritable. An important maintenance function of the PcG
and trxG protein complexes at CMMs might be to protect epigenetic marks
from erasure (Cavalli, 1999).
Dosage compensation ensures that males with a single X chromosome have the same amount of most X-linked gene products as females with two X chromosomes. In Drosophila, this equalization is achieved by a twofold enhancement of the level of X chromosome transcription in males, relative to each X chromosome in females. The products of at least five genes, maleless (mle), male-specific lethal 1, 2, and 3 (msl-1, msl-2, msl-3) and males absent on the first (mof), are necessary for dosage compensation. MOF transcript is found in larvae and adults of both sexes. The proteins produced by these genes form a complex that is preferentially associated with numerous sites on the X chromosome in the somatic cells of males, but not females. Binding of the dosage compensation complex to the X chromosome correlates with a significant increase in the presence of a specific histone isoform (histone 4 acetylated at lysine 16), on the chromosome. Experimental results and sequence analysis suggest that the mof gene encodes an acetyl transferase that plays a direct role in the specific histone acetylation associated with dosage compensation. The predicted amino acid sequence of MOF exhibits a significant level of similarity to several other proteins, including the human HIV-1 Tat interactive protein Tip60, the human monocytic leukemia zinc finger protein MOZ and the yeast silencing proteins SAS3 and SAS2. Also studied has been the role played by the various components of the complex in the targeting of MOF to the X chromosome. To this end, indirect cytoimmunofluorescence was used to monitor the binding of these components in males carrying either complete or partial loss-of-function mutations as well as in XX individuals in which formation of the dosage compensation complex has been induced by genetic means (Gu, 1998 and Hilfiker, 1997).
While the link between acetylation of histone H4 at lysine 16 and the dosage compensated male X chromosome points to an involvement of the modification early
on, a causal relationship between the two phenomena has been difficult to established. It has been considered possible that the
same principle that promotes an increase in accessibility of genes on the male X chromosome to the transcription machinery may also increase the availability
of the nucleosome substrate to a ubiquitous acetyltransferase. In this scenario, H4 acetylation would not be causal to the increased expression of X-linked genes in
males, but both phenomena would profit from a common, yet unidentified cause. It has now been shown that acetylation of nucleosomes at H4 lysine 16 can lead to a
remarkable relief of nucleosomal repression (Akhtar, 2000). Mof has been demonstrate to act is a
histone acetyltransferase that acetylates chromatin specifically at histone H4 lysine 16. This acetylation relieves chromatin-mediated repression of transcription in vitro
and in vivo if Mof is targeted to a promoter by fusion to a DNA-binding domain. Acetylation of chromatin by MOF, therefore, appears to be causally involved in
transcriptional activation during dosage compensation.
Dosage compensation in Drosophila therefore presents a strong case for a direct role of H4 acetylation on gene
transcription in vivo (Akhtar, 2000).
Hyperacetylation of histone H4 can lead to an increased access of transcription factors to nucleosomal DNA, to an unfolding of the
nucleosomal fiber, and to activation of transcription on chromatin templates. However, hyperacetylation is an experimentally induced condition in cells that is not observed under physiological conditons. Histones are mainly monoacetylated in vivo, and
the determinant of functional states appears to reside in the site specificity of acetylation rather than the extent of the modification. This has been nicely illustrated by
the visualization of histone H4 on Drosophila polytene chromosomes: acetylation of H4 at lysine 16 paints the hyperactive X chromosome, while acetylation at lysine
12 is a hallmark of inactive heterochromatin. Site-specific acetylation may conceivably affect interactions of the histone H4 N terminus with
either vicinal nucleosomes or nonhistone proteins. It is likely that these interactions may lead to alterations of the folding of the nucleosomal fiber and degree of
chromatin compaction. Interactions of the H4 N-terminal amino acids K16-N25 with the H2A/H2B heterodimer occur in the nucleosome crystal, but it is unclear yet whether these would contribute to the folding of a physiological chromatin fiber. The quest for interacting factors with potential to
discriminate between particular histone isoforms is ongoing, with no results reported to date (Akhtar, 2000 and references therein).
A cDNA fragment containing the putative Mof catalytic domain (aa 518 to
827) was expressed and it was determined that the recombinant peptide can acetylate Drosophila histones with a preference for histone H4. This
pattern is similar to that for a related yeast protein, Esa1p. Active full-length Mof could not be expressed, Mof was isolated as a component of a partially purified MSL complex. Tissue culture cells were used for the initial characterization of the MSL complex. S2 cells are male, based
on the following criteria: they do not express the Sxl (Sex-lethal) gene product, which is necessary for female differentiation, and they express Msl2, a limiting
component of the dosage compensation machinery whose synthesis is prevented by Sxl. S2 cells can be stably transfected, allowing the use of
commercially available antibodies recognizing epitope tags. Transient transfection of S2 cells with Msl2 tagged at its carboxy terminus with the HA epitope reveals
that the localization of the HA epitope is coincident with the location of endogenous Mof. After selection with hygromycin, most cells exhibit HA staining
on the male X chromosome, the location of which is revealed by antibodies to H4Ac16 (Smith, 2000).
Immunoprecipitation of nuclear extracts from Msl2-HA cells with the 12CA5 (anti-HA) antiserum results in the same proteins as those obtained from S2 cells
with an Msl1 antiserum. In salivary gland nuclei, Mle is released from the male X chromosome with RNase treatment. Furthermore, the roX1 and roX2 RNAs are found along the X
chromosome with a distribution that mimics that of the MSL complex. Therefore, attempts were made to obtain a partially purified complex
containing Mle and a roX RNA and to see whether the presence of either of these components depended on the other. 'RNA-friendly' conditions were developed to increase the chances of purifying Mle and roX RNA-containing complex. The method involved a cell line
expressing Flag-tagged MSL3 and sonication under low-salt conditions, immunoprecipitation with Flag antibodies followed by peptide elution, and a second
immunoprecipitation with either an MSL antibody or with the corresponding preimmune serum. By using this two-step procedure, a faint band was detected by silver
staining that corresponds to Mle protein. Clear enrichment of Mle was seen in the Msl1 immunoprecipitate relative to the preimmune serum.
However, following a brief treatment with 0.4 M NaCl, the Mle levels were significantly reduced (Smith, 2000).
To determine if roX RNAs are expressed in S2 cells, Northern blot analysis was performed and it was observed that roX2, but not roX1, is expressed in these cells, consistent with the observation that roX1 is dispensable in flies. The size of the major roX2 transcript observed by Northern analysis was ~ 600 nucleotides. To test if roX2 RNA is present in the Mle-containing immunoprecipitates, RNA was extracted from the
immunoprecipitation pellets and a RT-PCR was performed with roX2-specific primers in the linear range. The results show a clear enrichment of roX2 RNA in the
immune over the preimmune serum precipitates (Smith, 2000).
The MSL complex specifically acetylates lysine 16 of histone H4. When MSL-containing immunoprecipitates were incubated with nucleosomal substrates,
significant acetyltransferase activity toward histone H4 was detected. Msl1 immunoprecipitates from S2 nuclear extracts and 12CA5 immunoprecipitates
from Msl2-HA nuclear extracts contain H4-specific acetyltransferase activity, while control immunoglobulin G or 12CA5 immunoprecipitates from S2 cells do not.
To demonstrate that the acetyltransferase activity of the MSL complex is ascribable to Mof, complexes were purified containing either wild-type Mof or a protein
produced by the mutant allele mof1. This allele is a point mutation resulting in a glycine-to-glutamic acid replacement at the most highly conserved residue of the
acetyl-CoA binding domain (G691E). Wild-type Mof-HA or G691E Mof-HA were overexpressed in S2 cells and immunoprecipitated with anti-HA
antibodies to obtain complexes with only transfected Mof fusion proteins. Immunoprecipitates from G691E cells have markedly reduced acetylation, consistent with
the conclusion that Mof is the sole acetyltransferase in the MSL complex (Smith, 2000).
Given the specificity of the MSL complex toward H4, it was of intereset to determine which particular lysines were acetylated. When acetylated histones were separated by
acid-urea gel electrophoresis, predominantly monoacetylated H4 was detected. A similar acid-urea gel was blotted to PVDF, and the mono-acetylated
band was subjected to microsequencing. Counts were found at lysine 16, while other potential acetylation sites (at position 5, 8, or 12) were unlabeled. This
result provides a causative link between the presence of histone H4 acetylated at lysine 16 and the MSL complex on the X chromosome in Drosophila males (Smith, 2000).
Dosage compensation works to heighten the activity of the single X chromosome in males. This heightened expression of the X chromosome in males is accomplished through the action of male-specific lethal (MSL) proteins. Immunostaining of chromosomes shows that the MSL proteins are associated
with all female chromosomes at a low level but are sequestered to the X chromosome in males.
Histone-4 Lys-16 acetylation follows a similar pattern in normal males and females, being higher on the
X and lower on the autosomes in males than in females. However, the staining pattern of acetylation
and the mof gene product, a putative histone acetylase, returns to a uniform
genome-wide distribution as found in females and in males that are mutant for the msl gene. Gene expression on the autosomes correlates with the
level of histone-4 acetylation. With minor exceptions, the expression levels of X-linked genes are
maintained with either an increase or decrease of acetylation, suggesting that the MSL complex
renders gene activity unresponsive to H4Lys16 acetylation. Evidence has also been found for the presence
of nucleation sites for the association of the MSL proteins with the X chromosome rather than individual
gene binding sequences (Bhadra, 1999).
Mutations in Drosophila ISWI, a member of the SWI2/SNF2 family of chromatin remodeling ATPases, alter the global architecture of the male X chromosome. The transcription of genes on this chromosome is increased 2-fold relative to females due to dosage compensation, a process involving the acetylation of histone H4 at lysine 16 (H4K16). Blocking H4K16 acetylation suppresses the X chromosome defects resulting from loss of ISWI function in males. In contrast, the forced acetylation of H4K16 in ISWI mutant females causes X chromosome defects indistinguishable from those seen in ISWI mutant males. Increased expression of MOF, the histone acetyltransferase that acetylates H4K16, strongly enhances phenotypes resulting from the partial loss of ISWI function. Peptide competition assays have revealed that H4K16 acetylation reduces the ability of ISWI to interact productively with its substrate. These findings suggest that H4K16 acetylation directly counteracts chromatin compaction mediated by the ISWI ATPase (Corona, 2002).
The regulatory elements of the hsp26 promoter are well-known from in vivo and in vitro studies. A proximal regulatory element includes the TATA box, proximal heat shock element (HSE) and an adjacent GAGA element, while a distal regulatory site corresponds to the distal HSE and GAGA binding sites. Monitoring the ability of the transcription machinery to associate with the promoter in chromatin, it was found that Heat shock factor, a crucial regulator of heat shock gene transcription, profits most from histone acetylation. Templates with mutated hsp26 promoters were assembled into control and acetylated chromatin and analysed for their transcription potential. A template bearing only the TATA box supports a very low level of transcription, even in the absence of chromatin; there is no discernible transcription from chromatinized templates even after prolonged exposure. Addition of the proximal HSEs however, results in significantly increased transcription from both mock assembled and chromatinized templates, confirming the important role of the activator HSF. This minimal promoter, containing only proximal HSEs and the TATA box, clearly shows increased transcription from the acetylated template. The addition of GAGA elements to the promoter enhances transcription significantly, but to a similar degree on control and acetylated chromatin templates. Interestingly, the GAGA elements do not increase transcription in the mock assembled control, confirming that GAGA factor is involved in overcoming chromatin-mediated transcriptional repression, although the mechanism employed does not profit from histone acetylation. These results suggest that the HSE and TATA box are the significant sequence elements for the increased transcription observed in acetylated chromatin. Thus histone acetylation can modulate activator access to their target sites in chromatin, and provide a causal link between histone acetylation and enhanced transcription initiation of RNA pol II in chromatin (Nightingale, 1998).
Posttranslational modifications of the N-terminal tails of the core
histones within the nucleosome particle are thought to act as signals
from the chromatin to the cell for various processes. The experiments
presented here show that the acetylation of histones H3 and H4 in
polytene chromosomes does not change during heat shock. In contrast,
the global level of phosphorylated H3 decreases dramatically during a
heat shock, with an observed increase in H3 phosphorylation at the heat
shock loci. Additional experiments confirm that this change in
phosphorylated H3 distribution is dependent on functional heat shock
transcription factor activity. These experiments suggest that H3
phosphorylation has an important role in the induction of transcription
during the heat shock response (Nowak, 2000).
The acetylation of the N-terminal tails is the best-studied
modification of the core histones. Several transcription factors, such
as GCN5, and the TAFII250 subunit of TFIID, as well as subunits of the RNA polymerase complex show intrinsic histone
acetyltransferase (HAT) activity, which suggests a potential role for
histone acetylation in either the activation or maintenance of
transcription. The acetylation of the N-terminal tail domains of core
histones H3 and H4 at various lysine residues is essential for the
normal implementation of various cellular processes, such as
promoter-transcription factor association, gene transcription, and
dosage compensation (Nowak, 2000 and references therein).
Phosphorylation of serine 10 of the N-terminal arm of histone H3 has
been shown to be essential for proper mitotic chromosomal condensation and segregation. In addition, recent studies have outlined the possibility that
histone H3 phosphorylation may have a role in the regulation of
transcription. Ser 10 H3 phosphorylation is found to rapidly increase
in quiescent cells during mitogenic stimulation,
as well as during immediate-early gene induction via the epidermal
growth factor (EGF)-signaling pathway. In
addition, recent experiments performed in vitro have suggested that
EGF-stimulated H3 phosphorylation may act as a signal for histone
acetyltransferase binding and subsequent acetylation of a particular
locus during transcription initiation (Nowak, 2000 and references therein).
Acetylation of core histones H3 and H4 at lysines 14 and 8, respectively, has been linked to gene transcription. In addition, deacetylation of core histones is thought to have a role in silencing specific loci. Because of the
near-total repression of cellular gene products during a heat shock, it might be expected that the distribution of acetylated H3 and H4 would
radically change during thermal stress in a manner reflective of the
transcriptional profile of the cell. Because acetylation of H3 at Lys 14 of the
N-terminal arm has been described as essential for transcription, the
distribution of acetylated H3 was examined by staining polytene chromosomes with an
antibody specific for Lys 14 acetylated histone H3.
Lys 14 acetylated H3 staining is observed at the puffs, which are
active sites of transcription in polytene chromosomes, and distributed throughout the
chromosomes in discrete bands before heat shock. One locus,
subdivision 62A, which becomes puffed during larval development in
response to ecdysone, is intensely labeled with the
Lys 14 acetylated H3 antibody. In addition, other
chromosomal subdivisions such as 89B display Lys 14 acetylated H3
staining but are not puffed before heat shock. The
chromosomal subdivision 93D, which is known to become puffed during
heat shock, is Lys 14 acetylated but not puffed before heat shock. Examination of polytene chromosomes from larvae that were
subjected to a 20-min heat shock shows that the 87A and 87C heat shock
puffs, which contain the hsp70gene cluster, are stained by the anti-Lys 14 acetylated antibody, although
the staining at these puffs appears to be less intense and rather
diffuse. This might not represent a reduction in the
level of acetylation, but rather a decrease in signal intensity due to
the large puffing at the heat shock loci. After heat shock, the overall
number of discrete stained bands does not appear to change significantly
and regions that were stained before heat shock, such as 89B, remain
acetylated. Loci with acetylated H3 staining that were
puffed before heat shock, such as 62A, are no longer puffed after heat shock but remain acetylated. The observation that the
heat shock genes are acetylated before heat shock, at a time when they
are not transcribed, and non-heat shock genes, which are not
transcribed during heat shock, are acetylated during heat shock,
suggests that the presence of Lys 14-acetylated H3 does not necessarily
denote an actively transcribed locus (Nowak, 2000).
Examination of H3 acetylation during EGF stimulation raises the
issue that antibodies against Lys 14 acetylated H3 may show decreased
recognition of their epitope when other modifications, such as
phosphorylation, coexist on the same histone tail. This problem can be overcome by using antibodies against histone H3 acetylated at lysines 9 and 14. To ensure that these results were not caused by this potential artifact, the distribution of hyperacetylated H3 was examined using antibodies against H3 acetylated at lysines 9 and 14 on the N-terminal tail before and after heat shock. The results suggest that the distribution of diacetylated H3 is similar to
the distribution of Lys 14 acetylated H3 before and after heat shock.
Diacetylated H3 staining appears to be more widespread than
monoacetylated staining, which is probably caused by the antibody's
recognition of acetylation of H3 at lysine 9. The intensity of staining
of the Lys 9,14-acetylated H3 antibody at several of the heat shock
puffs examined appears to be similar to that observed with the Lys
14-acetylated H3 antibody. These results suggest that the diffuse staining at the heat shock puffs is not an artifact attributed to the masking of the acetylated Lys 14 epitope by Ser 10 phosphorylation (Nowak, 2000).
H4 acetylation was also examined using antibodies specific for Lys
8-acetylated histone H4 to stain polytene chromosomes isolated from third instar larvae. The distribution of Lys 8 acetylated histone H4 is similar to that of
acetylated H3, with H4 acetylation observed in discrete bands in
nonpuffed regions, such as subdivision 89B, and at ecdysone-induced
puffed regions, such as 62A, before heat shock. Chromosomal
subdivisions 87A and 87C, which contain the hsp70 heat shock
genes, are acetylated before and after
heat shock. Similar to acetylated H3, heat shock does not
significantly affect the observed distribution of Lys 8 acetylated H4
in polytene chromosomes. Taken together, the above results
suggest that the acetylation state of H3 and H4 does not change
substantially during heat shock and that a gene locus can be acetylated
when it is not actively transcribed (Nowak, 2000).
The absence of a drastic change in H3 acetylation during heat shock
is rather surprising, given current models that indicate that H3
acetylation is a crucial step in transcription initiation. This would lead to the expectation that the heat shock loci would not be acetylated before heat shock and should become intensely acetylated during thermal stress. To determine if
other histone modifications occur during the heat shock response, whether changes in histone H3 phosphorylation occur after
temperature elevation was examined. Stimulation of quiescent cells with EGF leads to
rapid and transient phosphorylation of histone H3 at Ser 10 of the
N-terminal arm in vivo. This EGF-mediated
phosphorylation of H3 is targeted to a small subpopulation of total
histone H3 that is acetylated at the Lys 14 position. In addition, in vitro studies have shown that phosphorylated H3
may serve as an affinity-increasing substrate for HAT activity in H3
acetylation, which raises the possibility that phosphorylation may be
tied to transcription. If histone phosphorylation were implicated in transcription, then the distribution of
phosphorylated H3 might change in response to heat shock and would most
likely be localized primarily to the heat shock puffs while
disappearing from other loci after heat shock. Because histone H3
phosphorylation is a robust marker for mitotic cells, analysis of the distribution of phosphorylated H3 in polytene
chromosomes, rather than isolation of phosphorylated H3 from whole cell
extracts, allows for the examining of phosphorylation of H3 in
a nonmitotic environment. To examine whether the heat shock-induced puffs contain N-terminal phosphorylated H3 molecules, polytene chromosomes were stained with antibodies specific for Ser 10 phosphorylated histone H3. Before heat shock,
phosphorylated H3 staining is found in discrete bands throughout the
chromosomes, with the most intense staining observed in the naturally
occurring ecdysone-induced developmental puffs. After a 20-min heat shock at 37°C, phosphorylated H3 staining is not distributed throughout the
chromosomes but is instead concentrated at a few specific sites. The most prominent of these regions corresponds to chromosomal
divisions 63BC, 67B, and 87AC. These regions contain the hsp83
gene, the hsp22, hsp23, hsp26, and
hsp27 gene cluster, and hsp70 gene clusters,
respectively. These regions become reproducibly puffed during the heat shock response. Although in some chromosomes examined there are
several non-heat shock loci that remain slightly phosphorylated
during heat shock, the intensity of staining at these regions is much
lower than the staining observed at the heat shock loci (Nowak, 2000).
The regions of the chromosome where the heat shock genes are located do
not contain histone H3 phosphorylated at Ser 10 before heat shock. After temperature elevation, the only puffs that possess
phosphorylated histone H3 are the heat shock puffs. The appearance of
phosphorylated histone H3 in the heat shock puffs, accompanied by the
nearly complete reduction of staining at all other loci during heat
shock, leads to the conclusion that the presence of the Ser 10 phosphorylated isoform of histone H3 might be required for the
transcriptional activation of the heat shock genes (Nowak, 2000).
Induction of the heat shock genes and cessation of normal gene
expression is rapid and reproducible in response to heat shock. Transcription run-on assays reveal that after only 1 min at 37°C, the levels of many normal cellular gene transcripts have greatly diminished, with the heat shock gene
transcripts dominating the population of total mRNA in the cell. Following a heat
shock, the normal pattern of gene expression within the cell is
restored gradually over time. Therefore an examination was made of
the change in phosphorylated histone H3 staining over time during and
after heat shock, to determine whether or not the appearance of
phosphorylated H3 closely parallels the induction of transcription
of the heat shock genes and whether or not the non-heat shocked H3
distribution might be restored following recovery from heat shock. After only 1 min at 37°C, there is a noticeable change in the distribution of Ser 10 phosphorylated H3. The
level of global H3 phosphorylation decreases, with several regions
remaining intensely phosphorylated. Within 5 min of incubation at 37°C, many of the less intense regions of staining have disappeared. After 10 min at 37°C, the only remaining intense regions of
staining are those at the heat shock puffs. When larvae were
allowed to recover at room temperature from a 20-min heat shock at
37°C, H3 phosphorylation reappears in several non-heat shock loci
after 10 min of recovery. After 30 min of recovery from heat
shock, the number and distribution of loci that contained
phosphorylated H3 appears to be virtually indistinguishable from
normal (i.e., non-heat shocked) chromosomes. This restoration
of the normal (non-heat shocked) H3 phosphorylation pattern closely
mimics previously described restoration of normal gene expression in
cells experiencing thermal stress (Nowak, 2000).
During heat shock, the heat shock transcription factor (HSF) rapidly
trimerizes in solution, localizes to the heat shock loci, binds to heat
shock response promoter elements (HSEs), and induces the expression of
the heat shock gene products. The appearance of phosphorylated H3 at the heat shock loci could therefore be due to HSF recruitment of a specific histone kinase on
binding to the HSEs of the heat shock genes. To test this hypothesis,
the staining pattern of phosphorylated H3 was examined in polytene
chromosomes isolated from hsf4-mutant larvae, which
lack functional HSF at restrictive temperatures and do not respond to
thermal stress. Before heat shock, the
distribution of phosphorylated H3 in hsf4-mutant
chromosomes is similar to wild-type chromosomes, with staining
observed in discrete bands and at the developmental puffs. In contrast to wild-type chromosomes, histone H3 at the heat shock loci does not become phosphorylated in
hsf4-mutant chromosomes during heat shock, which
suggests that phosphorylation of histone H3 at the heat shock loci
depends on functional HSF activity. In addition, no H3 phosphorylation was detected in the rest of the genome during heat
shock in hsf4 mutants, suggesting that repression of
normal transcription and loss of H3 phosphorylation at non-heat shock
loci does not require the presence of an active HSF protein (Nowak, 2000).
To determine if the loss of the HSF transcription factor could also
alter the distribution of acetylated H3 and H4 during heat shock, acetylation of each of these histones was examined in
hsf4-mutant polytene chromosomes. The distribution
of Lys 14 acetylated histone H3 before and after heat shock in
hsf4 mutants was indistinguishable from the
wild-type distribution, with staining observed at both the
developmental puffs and nonpuffed regions. H3 acetylation
was observed at the 87A and 87C chromosomal subdivisions, which
normally are puffed during heat shock but these regions do not become
puffed in hsf4-mutant chromosomes.
Examination of acetylated H3 using antibodies for Lys 9- and Lys
14-acetylated H3 shows a pattern similar to that observed for the Lys
14 acetylated H3 antibody. In addition, H4 acetylation does
not change after heat shock in hsf4 mutants. Because the heat shock genes are not induced in
hsf4 mutants during thermal stress and because
hsf4-mutant chromosomes are acetylated, but not
phosphorylated after heat shock, it is concluded that H3 phosphorylation,
and not acetylation, depends on the presence of a functional heat shock
transcription factor (Nowak, 2000).
How might acetylation and phosphorylation of histones H3 and H4 work
together to promote transcription of a particular gene? The data
suggest that acetylated histones might define a particular locus that
is primed for possible phosphorylation and subsequent transcription. This acetylated locus would attract transcription factors that interact with the
acetylated residues on histones H3 and H4, known to be essential for
proper association of several transcription factors with their
promoters. Once bound to this locus, the transcription factor would then
recruit a particular histone, which phosphorylates Ser
10 of the N-terminal arm of histone H3. The most logical site
of phosphorylation would be an H3 molecule with a Lys 14 acetylated
N-terminal arm, a species that has been shown to exist in vivo. The presence of this dimodified H3 would define that locus as 'active' for transcription (Nowak, 2000).
There are several kinases known to localize to specific loci on
polytene chromosomes that phosphorylate H3 in vitro, such as JIL-1 on
the X chromosome and P-TEFb kinase at the heat shock
loci (Lis, 2000). This raises the possibility that the
specificity of a kinase for activation of a particular gene through H3
phosphorylation might be regulated by the specific transcription
factors that control expression of this gene. It has yet to be determined
whether phosphorylation of H3 is required for assembly of the RNA
polymerase II complex or if phosphorylation is a by-product of complex
formation and polymerase procession during transcription. If
phosphorylation of H3 were indeed the critical step in activating gene
transcription, then a reasonable hypothesis is that deactivation of a
particular gene would be dependent on either regulated or unregulated
phosphatase activity to remove the activating phosphate group from the
N-terminal tails of H3. The disappearance of phosphorylated H3 at
nontranscribing loci and appearance of phosphorylated H3 at actively
transcribing loci during heat shock suggests that a functional
transcription complex might actively maintain the phosphorylated state
of histone H3, which would be subject to ready dephosphorylation by
either passive or regulated phosphatase activity in a nontranscribing state (Nowak, 2000).
Activation and repression of transcription in eukaryotes involve changes in the
chromatin fiber that can be accomplished by covalent modification of the histone
tails or the replacement of the canonical histones with other variants. The histone
H2A variant of Drosophila melanogaster, Histone H2A variant (H2Av),
localizes to the centromeric heterochromatin, and it is recruited to an ectopic
heterochromatin site formed by a transgene array. His2Av behaves
genetically as a PcG gene and mutations in His2Av suppress
position effect variegation (PEV), suggesting that this histone variant is
required for euchromatic silencing and heterochromatin formation. His2Av
mutants show reduced acetylation of histone H4 at Lys 12, decreased methylation
of histone H3 at Lys 9, and a reduction in HP1 recruitment to the centromeric
region. Neither H2Av accumulation nor histone H4 Lys 12 acetylation is affected by
mutations in either Su(var)3-9 or Su(var)2-5. The results suggest an
ordered cascade of events leading to the establishment of heterochromatin,
requiring the recruitment of the histone H2Av variant followed by H4 Lys 12
acetylation as necessary steps before H3 Lys 9 methylation and HP1 recruitment
can take place (Swaminathan, 2005).
Recent results suggest that H3 trimethylated at Lys 27 facilitates Pc binding to
silenced regions and this modification is carried out by
the Enhacer of zeste [E(z)] protein present in the ESC-E(z) complex.
Since a reduction in Pc on polytene
chromosomes was observed in His2Av mutants, whether recruitment of the
ESC-E(z) complex is also impaired in these mutants was examined.
In wild type, E(z) can be observed at multiple sites throughout the genome.
The levels and localization of E(z) do not appear to be altered in the
His2Av810 mutant compared to wild type.
Whether H3 Lys 27 methylation is affected by mutations
in His2Av was examined. The levels and distribution of this modification appear to be
the same in polytene chromosomes from wild-type and His2Av810
mutant larvae. This result was confirmed by Western analysis,
which shows equal levels of H3 trimethylated at Lys 27 in wild-type and
His2Av810 mutant larvae. These
results suggest that H2Av is required upstream of Pc recruitment in the process
of Pc-mediated silencing. Since neither recruitment of the E(z) complex nor H3
Lys 27 methylation seem to be affected in His2Av mutants, H2Av
replacement might take place after H3 Lys 27 methylation and before Pc
recruitment. Alternatively, Pc repression might require at least two parallel
and independent pathways, one involving H2Av recruitment and a second one
leading to H3 Lys 27 methylation, both of which might be required for proper Pc
recruitment (Swaminathan, 2005).
Formation of heterochromatin requires deacetylation of H3 Lys 9 followed by
methylation of the same residue and recruitment of HP1.
The heterochromatin of Drosophila chromosomes is enriched in
dimethylated and trimethylated histone H3 in the Lys 9 residue.
To analyze the possible role of H2Av in heterochromatin assembly,
the localization was examined of H3 dimethylated at Lys 9 in polytene chromosomes
from larvae carrying a mutation in the His2Av gene. Antibodies against
histone H3 dimethylated in Lys 9 stain the pericentric heterochromatin in
wild-type larvae. Interestingly, polytene
chromosomes from His2Av810 mutants show a decrease in the
amount of methylated H3 Lys 9, whereas the
presence of Su(Hw), used as a control, is the same in chromosomes from wild-type
and His2Av810 mutant larvae.
Since modification of this residue is important for HP1 recruitment,
whether localization of HP1 in heterochromatin is also affected by
mutations in His2Av was examined. In wild-type larvae, HP1 localizes preferentially to
the pericentric heterochromatin of
the chromocenter, but accumulation of HP1 is dramatically reduced in the
His2Av810 mutant (Swaminathan, 2005).
To confirm these results, Western analyses of protein extracts
obtained from wild-type and His2Av mutant larvae was
carried out using antibodies against
HP1 and histone H3 dimethylated in Lys 9. The results show little or no
accumulation of histone H3 methylated in Lys 9, and lower levels of HP1 in the
His2Av810 mutant. Methylation of
histone H3 at the Lys 9 residue is carried out by the Su(var)3-9 histone
methyltransferase, and HP1 is encoded by the
Su(var)2-5 gene. In order to
ensure that the observed effects on the levels of HP1 or the methylation of H3
Lys 9 were not caused by alterations in transcription of Su(var)3-9 or
Su(var)2-5 due to the His2Av mutation, quantitative
RT-PCR analyses of RNA obtained from wild-type and
His2Av810 mutant third instar larvae were carried out . The results show that
there are no significant changes in the levels of Su(var)3-9 or HP1 RNAs in
His2Av810 mutant larvae when compared to wild type.
These results and those from immunocytochemistry
analyses confirm a role for H2Av in the methylation of H3 Lys 9 and subsequent
recruitment of HP1 (Swaminathan, 2005).
Based on the observed effects of His2Av mutations on H3 Lys 9 methylation
and HP1 recruitment, it appears that the presence of H2Av in heterochromatin
might be required prior to these two events. To confirm this hypothesis,
the pattern of H2Av distribution on polytene chromosomes from larvae
carrying mutations was examined in the Su(var)2-5 and Su(var)3-9 genes. In both
cases, H2Av localization appears normal, suggesting
that the presence of H2Av is required prior to H3 Lys 9 methylation and HP1
recruitment during the establishment of heterochromatin (Swaminathan, 2005).
Sporulation in Saccharomyces cerevisiae is a highly regulated process wherein a diploid cell gives rise to four haploid gametes. This shows that histone H4 Ser1 is phosphorylated (H4 S1ph) during sporulation, starting from mid-sporulation and persisting to germination, and is temporally distinct from earlier meiosis-linked H3 S10ph involved in chromosome condensation. A histone H4 S1A substitution mutant forms aberrant spores and has reduced sporulation efficiency. Deletion of sporulation-specific yeast Sps1, a member of the Ste20 family of kinases, nearly abolishes the sporulation-associated H4 S1ph modification. H4 S1ph may promote chromatin compaction, since deletion of SPS1 increases accessibility to antibody immunoprecipitation; furthermore, either deletion of Sps1 or an H4 S1A substitution results in increased DNA volume in nuclei within spores. H4 S1ph is present during Drosophila melanogaster and mouse spermatogenesis, and similar to yeast, this modification extends late into sperm differentiation relative to H3 S10ph. Thus, H4 S1ph may be an evolutionarily ancient histone modification to mark the genome for gamete-associated packaging (Krishnamoorthy, 2006).
Whether H4 S1ph and H3 S10ph also occur during male meiosis and spermatogenesis in was investigated Drosophila. In spermatocytes undergoing the first meiotic division, metaphase I chromosomes stained strongly against both H4 S1ph and H3 S10ph antibodies. The H4 S1ph signal was detected on meiotic chromosomes from prophase through telophase for both meiosis I and meiosis II cells. In contrast, H3 S10ph staining was only prominent in metaphase and decreased substantially in anaphase and telophase. The H3 S10ph signal was also not detected in prophase spermatocytes. Immunofluorescence staining of Drosophila male germ cells undergoing spermatid differentiation revealed certain parallels between differentiation of yeast spores and male gametes in the behavior of H4 S1ph and H3 S10ph. During Drosophila spermatogenesis, as in yeast sporulation, H4 S1ph persisted until late in the terminal differentiation stages, while H3 S10ph levels were strongly reduced by the time meiosis was completed. Thus, in round and early elongating haploid spermatids, nuclei stained brightly for H4 S1ph, while staining for H3 S10ph was nearly undetectable in the same cells. The H4 S1ph epitopes persisted in spermatid nuclei as cells grew flagella and elongated and were still detected in nuclei undergoing chromatin compaction and nuclear shaping. Staining with H4 S1ph antibody gradually diminished at the later stages of nuclear elongation and shaping and was not detected in mature spermatids awaiting individualization. Staining for H3 S10ph was not detected in elongating spermatid nuclei at any stage (Krishnamoorthy, 2006).
Thus, similarities in the processes of sporulation and spermatogenesis, particularly in the drastic reduction in nuclear volume in both processes, led to an examination of whether H4 S1ph might correlate with the timing of chromatin compaction during spermatogenesis. H4 S1ph extending well beyond the time that meiosis-associated H3 S10ph is reduced during Drosophila and mouse spermatogenesis. In these metazoans, H3 S10ph and H4 S1ph both occur during meiotic divisions and thus may play a role in chromosome condensation. However, following meiotic divisions, the H3 S10ph is dramatically lowered, while H4 S1ph persists during the early stages of the developing spermatids when the genome begins to be compacted. This is true in mouse, as H4 S1ph continues to be present beyond the meiotic divisions and begins to be reduced contemporaneously with replacement of histones by the highly basic transition proteins. Although it is not yet yet known whether H4 S1ph has a role in genome compaction in metazoans, the data indicate that H4 S1ph has an additional role beyond meiotic divisions, as is the case in yeast. One clear difference between H4 S1ph in lower eukaryotes compared with metazoans is the persistence of the mark in mature spores and elimination only after germination. Thus, while H4 S1ph may directly promote stable chromatin compaction in mature spores, its role in metazoans may help to compact the genome connected to histone replacement by basic transition proteins. While many histone modifications have been correlated with broad genomic mechanisms such as transcription and DNA repair, the role of only a few modifications has been elucidated in higher-level biological processes. In this case, a central biological process, gametogenesis, is critically controlled in yeast by a single histone modification. The similarities observed between yeast and metazoans in the persistence of H4 S1ph after the decline of H3 S10ph emphasizes its importance (Krishnamoorthy, 2006).
Drosophila Pr-Set7 function was characterized based on the the availability of a P-element disruption of the 5' UTR region of the corresponding gene. A mutation in Drosophila pr-set7 is lethal: second instar larval death coincides with the loss of Histone H4 lysine 20 methylation, indicating a fundamental role for PR-Set7 in development. Transcriptionally competent regions lack H4 lysine 20 methylation, but the modification coincides with condensed chromosomal regions on polytene chromosomes, including chromocenter and euchromatic arms. The Drosophila male X chromosome, which is hyperacetylated at H4 lysine 16, has significantly decreased levels of lysine 20 methylation compared to that of females. In vitro, methylation of lysine 20 and acetylation of lysine 16 on the H4 tail are competitive. Taken together, these results support the hypothesis that methylation of H4 lysine 20 maintains silent chromatin, in part, by precluding neighboring acetylation on the H4 tail (Nishioka, 2002).
A mammalian histone methyltransferase (HMT) has been identified that is specific for lysine 20 of histone H4. This enzyme, PR-Set7, resides as a single polypeptide and is highly specific for nucleosomal histones. It was also shown that methylation of H4-K20 is associated with silent, transcriptionally inactive regions within euchromatin. Methylation of histone H4-K20 may maintain this higher order chromatin structure by inhibiting the acetylation of histone H4-K16. Taken together, these studies help to shed light on mechanisms that regulate chromatin structure through a series of concerted enzymatic reactions that ultimately 'mark' functionally distinct chromatin domains (Nishioka, 2002).
To identify and analyze HMTs present in human cells that specifically methylate histone H4, nuclear extracts from HeLa cells were fractionated on several chromatographic resins. Fractions from the columns were assayed for HMT activity using as substrates either core histone polypeptide or mono- and oligo-nucleosomes, in the presence and absence of histone H1. The separation of proteins in the DEAE-cellulose flowthrough (unbound) fraction on a negatively charged column (phosphocellulose) resulted in the resolution of two HMT activities, each with a different substrate and histone specificity. The histone H3-specific activity was eluted from the column at a lower salt concentration and was able to methylate core histone polypeptides as well as oligonucleosomes. This activity was specific for the K9 residue of H3 and was identified as Suv39h1. The other major HMT activity was eluted from the phosphocellulose column at a higher salt concentration and exclusively methylated nucleosomal histone H4. Further separation of the H4-specific HMT on a gel filtration column demonstrated that the activity had an apparent native mass of approximately 70 kDa. The final step of the purification scheme, fractionation on a Heparin agarose column, showed that the H4 HMT activity correlated with the appearance of a single polypeptide of approximately 40 kDa. It was later found by gel-filtration analysis that the enzymatically active 40 kDa protein resides as a homodimeric complex (Nishioka, 2002).
The purified native enzyme was subjected to further analysis, in order to more clearly define its substrate specificity. Assays were conducted with known substrates for several previously characterized protein methyltransferases, and it was found that the newly purified enzyme was highly specific for nucleosomal histone H4. A reaction mixture that contained nucleosomal histone H4, 3H-labeled S-adenosyl methionine (SAM), and the purified enzyme was then subjected to Edman degradation, and this analysis demonstrated that the target site for methylation is lysine 20. Moreover, when an HMT assay was carried out using nucleosomes reconstituted with an H4 species that contained an alanine in place of a lysine at position 20 (K20A), the newly purified HMT was unable to methylate the substrate, demonstrating further that this enzyme is specific for H4-K20 (Nishioka, 2002).
Mass spectrometric analysis of peptides derived from the protein that coeluted with the nucleosomal H4-specific HMT activity allowed probes to be generated with which to isolate a full-length cDNA clone. cDNA sequence analysis revealed that the activity was encoded by a gene that is absent in lower eukaryotes but is present in worms, flies, and vertebrates. The cDNA sequence matched perfectly with a sequence deposited in GenBank referred to as PR/SET domain containing protein 07 (accession number AAL40879). For simplicity, the enzyme was termed PR-Set7 (Nishioka, 2002).
Because a PR-Set7 homolog is present in Drosophila as a gene product of CG3307 (see Figure S1A), and because methylation of H4-K20 can be detected in the fruit fly, Drosophila was chosen as a model system to analyze the biological significance of this modification. The catalytic SET domain of Drosophila pr-set7 is about 40% identical in amino acid composition to that of human PR-Set7 (Nishioka, 2002).
Drosophila and mammalian PR-Set7 specifically methylate lysine 20 of histone H4 exclusively within a nucleosomal context. Although histone proteins have long been recognized to be methylated at specific residues in vivo, the enzymes that catalyze the modification reaction and the functions of these modifications have only recently begun to be revealed. Prior to this study, the function(s) of lysine methylated histone H4 was obscure, but was largely believed to be associated with transcriptionally active rather than repressed genes. However, this study has established that methylated H4-K20 is associated with silent chromatin. In support of the 'histone code hypothesis' methylation at H4-K20 inhibits acetylation of H4-K16 and vice versa. Consistent with the notion that an enzyme that alters the establishment of silent chromatin should have a tremendous impact on gene expression, these studies establish that the absence of methyl H4-K20 in vivo impairs the development and viability of a multicellular organism. Based upon the available evidence, the view is favored that a lack of, or diminishment of, H4-K20 methylation may alter patterns of gene expression, by perturbing a generally repressive, higher order chromatin structure that critically depends upon H4-K20 methylation (Nishioka, 2002).
The enzymatic activity of PR-Set7 is contained within a single polypeptide of ~40 kDa that appears to exist as a homodimer, because the native protein elutes from a gel filtration column with an apparent mass of ~70 kDa. The results demonstrate that PR-Set7 is an H4-K20-specific HMT, since the enzyme does not methylate any other residue on histone H4 or on any other histone. In agreement with previous studies demonstrating that the SET domain can be a signature for lysine-HMTs, PR-Set7 contains a SET domain, and a single substitution of a conserved arginine to glycine within the SET domain abolishes its enzymatic activity. Interestingly, PR-Set7 is devoid of the Pre- and Post-SET domains, demonstrating that these domains, although important for the functions of other HMTs, are not absolutely required for HMT activity. PR-Set7 is highly specific for nucleosomes, since no activity could be demonstrated when histones were used as a substrate. This lack of activity on nonnucleosomal histones is not likely to be due to the absence of the Pre- and Post-SET domains, because an HMT has been isolated with specificity for H3-K4 that exclusively methylates free histones and lacks both of these domains (Nishioka, 2002).
The establishment and maintenance of mitotic and meiotic stable (epigenetic) transcription patterns is fundamental for cell determination and function. Epigenetic regulation of transcription is mediated by epigenetic activators and repressors, and may require the establishment, 'spreading' and maintenance of epigenetic signals. Although these signals remain unclear, it has been proposed that chromatin structure and consequently post-translational modification of histones may have an important role in epigenetic gene expression. The epigenetic activator Ash1 has been shown to be a multi-catalytic histone methyl-transferase (HMTase) that methylates lysine residues 4 and 9 in H3 and 20 in H4. Transcriptional activation by Ash1 coincides with methylation of these three lysine residues at the promoter of Ash1 target genes. The methylation pattern placed by Ash1 may serve as a binding surface for a chromatin remodelling complex containing the epigenetic activator Brahma (Brm), an ATPase, and inhibits the interaction of epigenetic repressors with chromatin. Chromatin immunoprecipitation indicates that epigenetic activation of Ultrabithorax transcription in Drosophila coincides with trivalent methylation by Ash1 and recruitment of Brm. Thus, histone methylation by Ash1 may provide a specific signal for the establishment of epigenetic, active transcription patterns (Beisel, 2002).
Acetylation/de-acetylation, ubiquitination and methylation of histones (H1, H2A, H2B, H3, H4) have been correlated with the activation and silencing of transcription. Histone methylation occurs predominantly at arginine and lysine residues in the amino-terminal tails of H3 and H4. Arginine methylation mediates transcriptional activation by hormone receptors and probably other chromatin-dependent processes. By contrast, methylation of K9 and K4 in H3 and K20 in H4 has been linked to transcriptionally inactive chromatin, and corresponding HMTases have been identified. Methylation of H3 K4 has also been detected in transcription-competent chromatin, but the functional link between histone methylation and activation has not been dissected (Beisel, 2002).
To identify HMTases that establish activation-specific methylation patterns, a biochemical screen was used that identified Ash1, a member of the trithorax group of epigenetic activators as an HMTase. Ash1 contains a SET domain -- the 'signature motif' of lysine-specific HMTases -- flanked by cysteine-rich regions (pre-SET and post-SET domains). To confirm that Ash1 has HMTase activity, the ability to methylate histones was assessed in recombinant Ash1 derivatives Ash1DeltaN (deleted N terminus) and Ash1(SET) (containing the pre-SET, post-SET and SET domains only). The Ash1 derivatives methylate H3 and, to a lesser extent, H4 in polynucleosomes and histone core octamers. By contrast, 'free' H3 and H4 were methylated to a lesser extent compared with nucleosomes, even though free histones were present at a fivefold excess over polynucleosomal histones or when supplemented with DNA. These results suggest that Ash1 methylates H3 and H4. Since Ash1 used in the described HMTase assays was purified from eukaryotic cells, the HMTase activity of Ash1 could result from an associated rather than intrinsic activity. To test this, Ash1DeltaN was subjected to protein transfer membrane assays that detect intrinsic enzymatic activities in proteins. Ash1(SET) was separated by SDS-polyacrylamide gel electrophoresis (PAGE), transferred electrophoretically onto polyvinylidene fluoride (PVDF) membrane, and denatured/re-natured. Reconstituted Ash1(SET) methylates H3 and H4, suggesting that Ash1 has intrinsic HMTase activity (Beisel, 2002).
To identify the target amino acid residue(s) of Ash1, radiolabelled H3 was subjected to Edman-degradation. Scintillation counting of the released amino acid fractions detected radiolabelling of H3 K4 and K9. To support this, the ability of Ash1DeltaN to methylate peptides consisting of amino acids 1-20 of H3 [H3(1-20)] was tested. Ash1DeltaN methylates the peptides H3(1-20), H3(1-20)K4 (which contains H3 K4 but leucine residues instead of lysine residues at positions 9, 14 and 18) and H3(1-20)K9 (which contains H3 K9 but leucine residues instead of lysine residues at positions 4, 14 and 18). By contrast, H3(1-20)L4/L9 peptides, which contain leucine residues at position 4 and 9 of H3, are not significantly methylated, indicating further that Ash1 methylates H3 K4 and K9. Owing to the weak radiolabelling, the target(s) of Ash1 in H4 could not be identified by Edman-degradation. Since H4 K20 is the only H4 residue being methylated in vivo, a monoclonal antibody was generated against dimethylated H4 K20 [anti-dim(H4-K20)] to investigate whether Ash1 methylates H4 K20. H4 that was free, in histone core octamers or polynucleosomes, was methylated by Ash1 and analysed by Western blot analysis. Anti-dim(H4-K20) antibody recognizes Ash1-methylated H4, but not un-methylated H4, indicating that Ash1 methylates H4 K20 (Beisel, 2002).
Single amino acid point mutations ash110 and ash121 abolish Ash1 activator function in Drosophila. The mutation in ash110 (N1458I) resides within the SET domain, and in ash121 (E1357K) in the pre-SET domain. To assess whether these mutations affect HMTase activity, recombinant proteins were expressed and purified containing one of these mutations (Ash1DeltaN10, Ash1DeltaN21) and a third mutant (Ash1DeltaN1142) whose mutation (H1459K) resides in the SET domain and abolishes HMTase activity of SUV39H1. HMTase assays revealed that the mutants do not significantly methylate H3 and H4, indicating that the mutations abolish HMTase activity and that both the pre-SET and SET domains of Ash1 contribute to HMTase activity and transcriptional activation by Ash1 (Beisel, 2002).
To assess whether the mutations in Ash1 specifically inactivate HMTase activity or cause a general functional inactivation, the ability of mutant Ash1DeltaN to bind the known interaction partner Trx was investigated. Ash1DeltaN and the three mutants can interacte with Trx in vitro, suggesting that the inability of mutant Ash1 proteins to methylate histones is based on a specific inactivation of HMTase activity (Beisel, 2002).
To investigate the effect of ash110 and ash121 on transcriptional activation by Ash1 in Drosophila, transgenic flies were used carrying the Ash1-dependent reporter gene N18/15, which contains a 4-kilobase (kb) regulatory element of the bxd region from the Ash1 target gene Ubx fused to the mini-white gene. Ash1 supports activation of N18/15 transcription in the Drosophila eye. By contrast, N18/15 expression is significantly reduced in ash110/ + or ash121/+ heterozygous flies. Since Ash121 and Ash110 lack HMTase activity in vitro, these results imply that HMTase activity contributes to transcriptional activation by Ash1 in vivo (Beisel, 2002).
To dissect the functional relationship between transcriptional activation and histone methylation by Ash1, transcriptional activation by Ash1 was reconstituted in Drosophila S2 cells. To monitor transcription in chromatin, S2 (BCAT5) cells were generated that carry the stable integrated reporter gene BCAT5, which contains five DNA-binding sites for the yeast activator Gal4, a core promoter and the bacterial cat gene. To recruit Ash1 to chromatin, Ash1 derivatives were fused to the Gal4 DNA-binding domain (amino acids 1-147) [Gal4(DBD)]. BCAT5 cells were transfected with plasmids expressing fusion proteins comprising Gal4(DBD) and either wild type or mutant Ash1DeltaN. Gal4(DBD)-Ash1DeltaN activates BCAT5 expression 20-fold, whereas HMTase-inactive Ash1DeltaN derivatives did not. These results support the hypothesis that HMTase activity of Ash1 mediates activation of transcription (Beisel, 2002).
To link transcriptional activation by Ash1 to histone methylation, crosslinked chromatin immunoprecipitation (XChIP), which detects protein-DNA interactions in vivo, was used. Crosslinked chromatin was isolated from BCAT5 cells expressing Gal4(DBD)-Ash1DeltaN, Gal4(DBD)-Ash1DeltaN10 or Gal4(DBD)-Ash1DeltaN21, and immunoprecipitated by antibodies recognizing dimethylated H3 K4, H3 K9 or H4 K20. Precipitated DNA was purified and the enhancer/promoter of target genes was detected by polymerase chain reaction. All three antibodies precipitate chromatin containing the BCAT5 enhancer/promoter from cells in which Gal4(DBD)-Ash1DeltaN activates transcription. Methylation of these lysine residues was detectable 500 bp upstream of the enhancer/promoter and at the 3'-end of the cat gene. In cells expressing Gal4(DBD)-Ash1DeltaN10, methylation of H3 K4 was undetectable, but weak methylation of H3 K9 and H4 K20 could be observed. This finding supports current models proposing that transcriptional repression correlates with methylation of H3 K9 and H4 K20. As, however, H3 K9 and H4 K20 methylation is enhanced at the transcriptionally active (active) reporter, transcriptional activation by Ash1 correlates with de novo methylation of not only H3 K4 but also H3 K9 and H4 K20 (Beisel, 2002).
Methylation of H3 K9 at the transcriptionally silent (silent) enhancer/promoter implies that BCAT5 might be associated with HP1, which binds methylated H3 K9. This was tested by XChIP using anti-HP1 polyclonal antibody. The antibody precipitated the BCAT5 enhancer/promoter from cells expressing HMTase-inactive Gal4(DBD)-Ash1DeltaN10. By contrast, the enhancer/promoter was only weakly precipitated from cells in which Gal4(DBD)-Ash1DeltaN activates reporter expression. These results suggest that HP1 binds the silent enhancer/promoter and is removed/relocated from the reporter by Ash1-mediated histone methylation (Beisel, 2002).
To investigate whether Ash1 methylates histones to activate transcription of a natural target gene, methylation of Ubx was monitored in BCAT5 cells. Ubx is not expressed in S2-cells but PCR with reverse transcription (RT-PCR) indicates that transiently expressed Gal4(DBD)-Ash1DeltaN activates expression of this gene in BCAT5 cells. XChIP experiments indicate that H3 K4 is not methylated and that H3 K9 and H4 K20 are only weakly methylated at the silent Ubx promoter. By contrast, methylation of all three lysine residues is significantly enhanced when Ash1 activates BCAT5 expression, indicating that transcriptional activation of Ubx by Ash1 coincides with methylation of H3 K4, K9 and H4 K20 (Beisel, 2002).
Genetic data indicate that Ash1 activates Ubx expression in imaginal discs of the third leg. Therefore, to investigate histone methylation by Ash1 in the natural context of the activator, the methylation pattern of Ubx was examined in third leg discs by XChIP. Crosslinked chromatin was prepared from third leg discs dissected from third instar larvae. Chromatin immunoprecipitations indicated methylation of H3 K4, K9 and H4 K20 at the Ubx promoter, suggesting that Ash1-mediated methylation of all three lysine residues coincides with epigenetic activation of Ubx transcription in Drosophila (Beisel, 2002).
On the basis of the result that methylated lysine residues facilitate or inhibit the binding of proteins, an investigation was carried out to determine whether the trivalent (H3 K4, K9 and H4 K20) methylation pattern placed by Ash1 attracts or repels proteins to establish epigenetic activation. The XChIP experiments in indicate that the trivalent methylation pattern removes/relocates HP1 from chromatin. To support this finding, the interaction was investigated of HP1 with methylated H3 peptides and histone core octamers that had been methylated by Ash1 or Drosophila SU(VAR)3-9, which methylates H3 K9. HP1 binds H3 K9-methylated peptides and histone core octamers, as well as H3 K4/K9-methylated peptides. By contrast, HP1 does not bind Ash1-methylated core octamers, suggesting that the trivalent methylation pattern inhibits the interaction of HP1 with chromatin (Beisel, 2002).
Protein-protein interaction assays using H3(1-20) peptides methylated at H3 K4, K9 or H3 K4 and K9 (H3 K4/K9), and Drosophila embryonic nuclear extract or recombinant proteins, resulted in the identification of three proteins that exhibit differential binding to methylated peptides. Two of these proteins -- the epigenetic repressor Polycomb (Pc) and Caf-1 p55, a subunit of different protein complexes involved in, for example, epigenetic repression -- bind H3 K9-methylated peptides and histone core octamers, but show significantly reduced binding to H3 K4- or H3 K4/K9-methylated peptides and trivalently methylated histone core octamers. Furthermore, protein-binding assays indicate that Brm and Moira (Mor) interact with H3 K4/K9-methylated peptides. In contrast, both proteins were not recruited to peptides methylated at H3 K4 or H3 K9. Brm and Mor are subunits of a SWI/SNF-like chromatin remodelling complex, suggesting that this complex, rather than individual proteins, is recruited to K4/K9-methylated H3. These results imply that the trivalent methylation pattern established by Ash1 facilitates or prevents the interaction of proteins with methylated H3 during epigenetic activation. To support this finding, XChIP was used to investigate the interaction of Brm and repressors with Ash1 target genes. These analyses indicate that Brm and Mor are present at the active but not at silent promoters of Ash1 target genes in cells or third leg imaginal discs. By contrast, the repressors were only detected at silent promoters. Thus, transcriptional activation by Ash1 may coincide with the recruitment of Brm and Mor and the extinction of repressor binding at the promoter of Ash1 target genes (Beisel, 2002).
Collectively, these data indicate that the epigenetic activator Ash1 activates transcription by methylation of H3 K4, K9 and H4 K20 at the promoter of target genes. This suggests that epigenetic activation and silencing, which has been linked to methylated H3 K9, may correlate with different histone methylation patterns. Each of the three lysine residues targeted by Ash1 can be individually methylated by specific HMTases, resulting in transcriptional repression and probably activation (H3 K4). Combining these three modifications results in a novel biological readout: epigenetic activation. Why does the trivalent modification pattern generated by Ash1 mediate epigenetic activation? The results indicate that each modification of the pattern fulfils a specific function. Methylation of H3 K4 prevents the interaction of repressors (Pc, p55) with Ash1 target genes. Methylation of H4 K20 in addition to H3 K4 and H3 K9 prevents the interaction of HP1 with chromatin. Inhibition of repressor binding is an important mechanism, as epigenetic activators and repressors are expressed together during Drosophila development. Finally, methylation of H3 K4 and H3 K9 generates an interaction surface for a chromatin-remodelling complex. These results imply that a specific functional interplay between the epigenetic activators Ash1 and Brm mediates epigenetic activation of transcription. Ash1 initially binds target genes and generates the trivalent histone methylation pattern, which subsequently recruits a Brm-containing chromatin-remodelling complex. The activity of this complex may contribute to the establishment of epigenetic active chromatin structures (Beisel, 2002).
Histone lysine methylation is a central modification to mark functionally distinct chromatin regions. In particular, H3-K9 trimethylation has emerged as a hallmark of pericentric heterochromatin in mammals. H4-K20 trimethylation is also focally enriched at pericentric heterochromatin. Intriguingly, H3-K9 trimethylation by the Suv39h HMTases is required for the induction of H4-K20 trimethylation, although the H4 Lys 20 position is not an intrinsic substrate for these enzymes. By using a candidate approach, Suv4-20h1 and Suv4-20h2 were identified as two novel SET domain HMTases that localize to pericentric heterochromatin and specifically act as nucleosomal H4-K20 trimethylating enzymes. Interaction of the Suv4-20h enzymes with HP1 isoforms suggests a sequential mechanism to establish H3-K9 and H4-K20 trimethylation at pericentric heterochromatin. Heterochromatic H4-K20 trimethylation is evolutionarily conserved, and in Drosophila, Suv4-20 is a novel position-effect variegation modifier. Together, these data indicate a function for H4-K20 trimethylation in gene silencing and further suggest H3-K9 and H4-K20 trimethylation as important components of a repressive pathway that can index pericentric heterochromatin (Schotta, 2004).
These data suggest H4-K20 trimethylation is a mark of silenced chromatin domains. Therefore whether this modification would indeed be important for gene silencing in well-described PEV models in Drosophila was investigated. A single, homozygous-viable P-element insertion (P{GT1}BG00814) into the third exon of Suv4-20 has been identified in the course of the Drosophila gene disruption project. H4-K20 trimethylation at polytene chromatin is nearly lost in homozygous mutant larvae, demonstrating that the P-element insertion (Suv4-20BG00814) represents a strong hypomorphic allele of Suv4-20. Because the Suv4-20 locus maps on the X chromosome, the classical PEV rearrangement In(1)wm4 cannot be used to analyze a potential modifier effect of Suv4-20. Therefore, another PEV rearrangement was analyzed that translocates a different marker, Stubble (Sb), close to pericentric heterochromatin (T(2;3)SbV). The dominant mutation Stubble induces short bristles, but heterochromatin-induced silencing of SbV results in wild-type (long) bristles. Homozygous Suv4-20BG00814 as well as control wild-type females were crossed to T(2;3)SbV males. In the progeny, the extent of SbV reactivation was determined as the ratio of short bristles (active SbV) to long bristles (inactive SbV). In males and females of the wild-type crosses, only 1%-2% of bristles show a Sb phenotype, indicating that SbV is largely inactivated. In contrast, SbV becomes derepressed in the progeny of Suv4-20BG00814 flies, because now ~25% of the bristles are short. This result classifies Suv4-20 as a dominant PEV modifier and further indicates a functional role for Suv4-20-dependent H4-K20 trimethylation in gene silencing (Schotta, 2004).
The ATPase ISWI can be considered the catalytic core of several multiprotein
nucleosome remodeling machines. Alone or in the context of nucleosome remodeling
factor [the chromatin accessibility complex (CHRAC), or ACF] ISWI catalyzes a
number of ATP-dependent transitions of chromatin structure that are currently
best explained by its ability to induce nucleosome sliding. In addition, ISWI
can function as a nucleosome spacing factor during chromatin assembly, where it
will trigger the ordering of newly assembled nucleosomes into regular arrays.
Both nucleosome remodeling and nucleosome spacing reactions are mechanistically
unexplained. As a step toward defining the interaction of ISWI with its
substrate during nucleosome remodeling and chromatin assembly a set
of nucleosomes lacking individual histone N termini were generated from recombinant histones. The conserved N termini (the N-terminal tails) of histone H4 were found to be essential
to stimulate ISWI ATPase activity, in contrast to other histone tails.
Remarkably, the H4 N terminus, but none of the other tails, is critical for
CHRAC-induced nucleosome sliding and for the generation of regularity in
nucleosomal arrays by ISWI. Direct nucleosome binding studies did not reflect a
dependence on the H4 tail for ISWI-nucleosome interactions. It is concluded that the H4 tail is critically required for nucleosome remodeling and spacing at a step subsequent to interaction with the substrate (Clapier, 2001).
The chromatin accessibility complex (CHRAC) was originally defined biochemically as an ATP-dependent 'nucleosome remodelling'
activity. Central to its activity is the ATPase ISWI, which catalyses the transfer of histone octamers between DNA segments in cis. In
addition to ISWI, four other potential subunits were observed consistently in active CHRAC fractions. The p175 subunit of CHRAC has been identified as Acf1, a protein known to associate with ISWI in the ACF complex. Interaction of Acf1 with ISWI enhances the efficiency of nucleosome sliding by an order of magnitude. Remarkably, it also modulates the nucleosome remodelling activity of ISWI
qualitatively by altering the directionality of nucleosome movements and the histone 'tail' requirements of the reaction. The Acf1-ISWI
heteromer tightly interacts with the two recently identified small histone fold proteins CHRAC-14 and CHRAC-16. Whether topoisomerase II is an integral subunit
has been controversial. Refined analyses now suggest that topoisomerase II should not be considered a stable subunit of CHRAC. Accordingly, CHRAC can be
molecularly defined as a complex consisting of ISWI, Acf1, CHRAC-14 and CHRAC-16 (Eberharter, 2001).
A heterodimeric complex of Acf1 and ISWI previously had been termed 'ACF'. In this context, Acf1 significantly increases the activity of ISWI in
chromatin assembly. Since Acf1 has been identified as a component of CHRAC, the impact of Acf1 on ISWI-induced nucleosome sliding was examined. The directionality of nucleosome sliding differs depending on whether the reaction is catalysed by ISWI alone or by CHRAC. Flag-tagged ISWI and Acf1 were expressed from baculovirus vectors in insect cells, affinity purified and assayed for nucleosome sliding. In agreement with previous results, catalytic amounts (2-3 fmol) of ISWI move a mononucleosome from the center of a 248 bp rDNA fragment to the fragment end. No mobility was observed when the end-positioned nucleosome is exposed to ISWI. In contrast to the movement generated by ISWI, CHRAC catalyses nucleosome sliding from the end to the center of the DNA fragment. Strikingly, CHRAC-type directionality of nucleosome sliding is also obtained if Acf1 is added to ISWI, either after separate expression or by co-expression of both proteins in Sf9 cells. While Acf1 alone is inactive for nucleosome sliding, it boosts ISWI activity by at least an order of magnitude
such that 10-fold lower enzyme concentrations (0.3-0.5 fmol) are required for nucleosome mobilization. Most importantly, Acf1 changes the directionality of
sliding such that end-positioned nucleosomes move to central positions (Eberharter, 2001).
In order to determine whether Acf1 has an additional effect on the kinetics of nucleosome mobility under these conditions, a time course of
nucleosome mobility was performed. The amounts of enzymes were chosen such that complete mobilization of the nucleosome was expected after 90 min (10-fold less
ACF than ISWI). At any given time point throughout the reaction, the ratio of nucleosomes that had been mobilized to those that had not moved was determined.
Nucleosome movement in the two reactions proceeds with similar speed, indicating that ACF is about an order of magnitude more efficient in nucleosome
mobilization than ISWI alone. This could be explained most readily if Acf1 stimulates the ATPase activity of ISWI. To determine whether this was the case, the enzymes were compared in standard ATPase assays. ISWI alone shows a robust (7-fold) nucleosome stimulation of ATPase. This response to a
nucleosomal structure remains unaltered if Acf1 is added, either after separate expression or through co-expression. While Acf1 alone does not show any sign of ATPase activity, it also does not stimulate the ATPase of ISWI significantly (7-fold stimulation over the free DNA level in all cases) (Eberharter, 2001).
Deletion of the H4 N-termini completely abolishes the ability of CHRAC to slide nucleosomes, whereas removal of any other histone tail has
only minor effects. In contrast, ISWI-induced sliding not only requires the histone H4 N-termini (like CHRAC), but is also impaired if any of
the other tails are deleted. Since Acf1 modulates the directionality of nucleosome sliding to resemble that of CHRAC, the histone
tail dependence of ACF-induced nucleosomal sliding was tested. As expected, deleti
