TBP-associated factor 1: Biological Overview | Evolutionary Homologs | Regulation | References

Gene name - TBP-associated factor 1

Synonyms - TAFII250, TBP-associated factor 250kD

Cytological map position - 84A2--84A3

Function - TBP associated factor

Keywords - general transcription factor

Symbol - Taf250

FlyBase ID:FBgn0010355

Genetic map position - 3-[47.1]

Classification - Taf250 - kinase and histone transacetylase

Cellular location - nuclear



NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Lienard, M. A., Araripe, L. O. and Hartl, D. L. (2016). Neighboring genes for DNA-binding proteins rescue male sterility in Drosophila hybrids. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 27357670
Summary:
Crosses between closely related animal species often result in male hybrids that are sterile, and the molecular and functional basis of genetic factors for hybrid male sterility is of great interest. This study reports a molecular and functional analysis of HMS1, a region of 9.2 kb in chromosome 3 of Drosophila mauritiana, which results in virtually complete hybrid male sterility when homozygous in the genetic background of sibling species Drosophila simulans. The HMS1 region contains two strong candidate genes for the genetic incompatibility, agt and Taf1. Both encode unrelated DNA-binding proteins, agt for an alkyl-cysteine-S-alkyltransferase and Taf1 for a subunit of transcription factor TFIID that serves as a multifunctional transcriptional regulator. The contribution of each gene to hybrid male sterility was assessed by means of germ-line transformation, with constructs containing complete agt and Taf1 genomic sequences as well as various chimeric constructs. Both agt and Taf1 contribute about equally to HMS1 hybrid male sterility. Transgenes containing either locus rescue sterility in about one-half of the males, and among fertile males the number of offspring is in the normal range. This finding suggests compensatory proliferation of the rescued, nondysfunctional germ cells. Results with chimeric transgenes imply that the hybrid incompatibilities result from interactions among nucleotide differences residing along both agt and Taf1. The results challenge a number of preliminary generalizations about the molecular and functional basis of hybrid male sterility, and strongly reinforce the role of DNA-binding proteins as a class of genes contributing to the maintenance of postzygotic reproductive isolation.
BIOLOGICAL OVERVIEW

Initiation of transcription (the synthesis of messenger RNA) is carried out by a large protein complex, containing at least 30 proteins. As the understanding of the existence and functioning of such a large protein complex has developed, so has the realization that transcriptional initiation is highly regulated, and that each of these 30 protein components plays an important role in mRNA synthesis. The discussion of Taf1 (below) presumes some familiarity with the proteins involved in the initiation of transcription. If you are unfamiliar with these proteins, you might want first refer to RNA polymerase and general transcription factors before continuing to read about TAF1.

If one were to short list proteins according to the significance of their contribution to transcriptional initiation, Taf1 would be somewhere near the top of the list. Taf1 is the largest of the eight or nine transcription associated factors (TAFs) in Drosophila. TAFs are components of TFIID, a target of many transcriptional activators whose interactions with TFIID may enhance promoter function. In the most general case, the orderly process of transcription initiation begins with TFIID recognizing and binding tightly (via TATA binding protein or TBP, a component of TFIID) to the TATA box, a conserved sequence in many promoters. Taf1 has been shown to interact directly with TATA binding protein; Taf1 also harbors kinase activity (Dikstein, 1996) that targets itself (autophosphorylation) and TFIIF, another component of the initiation complex. TFIIF stimulates transcriptional elongation, and is a homolog of the bacterial sigma factor. RNA polymerase II cannot stably associate with a TFIID and TFIIB assembly at the promoter and must be escorted to the promoter by TFIIF (Orphanides, 1996). Therefore TFII250 regulates a protein (TFIIF) that chaperones (accompanies) RNA polymerase to the promoter.

What is the role of Taf1 in transcriptional activation of promoters? Central to eukaryotic transcriptional regulation is how TFIID gains access to a chromatin template and how a stable association is maintained with the chromosomal environment. Drosophila Taf1 and human and yeast homologs possess histone acetyltransferase (HAT) activity. Acetylation of histones is implicated in two related processes in eukaryotes: (1)acetylation is involved in the process of histone assembly into nucleosomes, and 2) histone acetylation plays a positive role in promoting access of transcription factors to nucleosomal DNA. These two processes are discussed more fully at the Histone 4 site. Thus Taf1, by means of a histone transacetylase function, has the potential to modify the protein bound environment of eukaryotic DNA (by acetylation), presumably modifying the access of chromatin, at or near core promoters, to other factors involved in transcriptional regulation (Mizzen, 1996).

Studies in unicellular systems indicate that TAF250 is required for progression through G1/S of the cell cycle and repression of apoptosis. These in vivo studies have been extended by determining the developmental requirements for TAF250 in a multicellular organism, Drosophila. TAF250 mutants were isolated in a genetic screen that also yielded TAF60 and TAF110 mutants, indicating that TAFs function coordinately to regulate transcription. Null alleles of TAF250 are recessive larval lethal. However, combinations of weak loss-of-function TAF250 alleles survive to adulthood and reveal requirements for TAF250 during ovary, eye, ocelli, wing, bristle, and terminalia development as well as overall growth of the fly. These phenotypes suggest roles for TAF250 in regulating the cell cycle, cell differentiation, cell proliferation, and cell survival. Finally, molecular analysis of TAF250 mutants reveals that the observed phenotypes are caused by mutations in a central region of TAF250 that is conserved among metazoan organisms. This region is contained within the TAF250 histone acetyltransferase domain, but the mutations do not alter the histone acetyltransferase activity of TAF250 in vitro, indicating that some other aspect of TAF250 function is affected. Because this region is not conserved in the yeast TAF250 homolog, TAF145, it may define an activity for TAF250 that is unique to higher eukaryotes (Wassarman, 2000).

TAF250 (also designated TAF230 in Drosophila, CCG1 in humans, and TAF145/135 in yeast), is required for progression through the G1/S boundary of the cell cycle. Mammalian or yeast cells carrying temperature-sensitive alleles of TAF250 arrest in G1 at the nonpermissive temperature. Furthermore, following growth arrest, mammalian cells undergo apoptosis. In addition to serving as a scaffold on which other TAFs and TBP are assembled, TAF250 possesses enzymatic, promoter recognition, TBP regulatory, and coactivator activities. Transcriptional defects observed in these cells at the nonpermissive temperature presumably result from elimination of one or more TAF250 activities (Wassarman, 2000 and references therein).

TAF250 contains two independent protein kinase domains and a histone acetyltransferase (HAT) domain. The kinase domains can autophosphorylate as well as transphosphorylate, in vitro, RAP74, the largest subunit of TFIIF. The HAT domain can acetylate lysine residues in vitro, in the N-terminal tails of histones H3 and H4. Because acetylation of histones induces changes in chromatin structure, the HAT activity of TAF250 may provide a mechanism for TFIID to access transcriptionally repressed chromatin. The TAF250 bromodomains may contribute to this process because bromodomains have been shown to bind the N terminus of histone H4 and acetylated Lys residues (Wassarman, 2000 and references therein).

Mutations in Drosophila TAF250 have been identified in a genetic screen that is sensitive to transcription levels. The availability of loss-of-function mutations in TAF250 has allowed for a determination of how Drosophila development is affected by the absence of this broadly acting transcriptional regulator; such mutations allow for the definition of a protein domain that is critical for the function of TAF250 in vivo. Changes in the transcription profile of a cell occur in response to signals mediated by the Ras GTPase. Much work has focused on the gene-specific enhancer-binding proteins that are direct targets of the Ras signal, but components of the transcriptional machinery that interact with enhancer-binding proteins presumably play equally important roles in regulating transcription. Consistent with this proposal, mutations in subunits of TFIIA and TFIID interfere with the ability of Ras1 to specify cell fate decisions during Drosophila eye development. Mutations in TAF60 and TAF110 were isolated in a genetic screen as suppressors of the rough eye phenotype caused by misexpression of a constitutively active form of Ras1 (Ras1V12) under transcriptional control of the sev enhancer and promoter elements (sev-Ras1V12). Genetic interaction studies were then used to categorize the isolated modifiers into two general groups: Class I, genes that function directly in the Ras1 signaling pathway, and Class II, genes that function to transcriptionally regulate the sev-Ras1V12 transgene or downstream gene targets of the pathway. Class I groups have subsequently been shown to include protein kinases that function in a general Ras1 signaling cassette and the only Class II groups characterized thus far, SR3-4B and SR3-3, correspond to TAF60 and TAF110. Thus, it appears likely that additional Class II groups (SR3-2, SR3-4A, SR3-5, SR3-7, and SR3-8) will encode transcriptional regulators. This study focuses on SR3-5 (Wassarman, 2000).

The SR3-5 locus encodes Drosophila TAF250. Searches of the Berkeley Drosophila Genome Project (BDGP) database identified a mutant line, EP(3)0421, in which a P-element is inserted within exon 1 of TAF250, 55 bp downstream of the translation start site. To determine if TAF250 expression is affected in EP(3)0421 flies, ovary extracts were prepared from wild-type and EP(3)0421 heterozygous and homozygous flies and probed with a TAF250 antibody. Relative to TAF110, TAF250 protein expression is drastically reduced in EP(3)0421 homozygous flies. EP(3)0421 is homozygous viable and complements the lethality of SR3-5S-136 and SR3-5S-625, but it only partially complements SR3-5XS-2232 (Wassarman, 2000).

To determine whether the phenotypes of EP(3)0421-containing flies are caused by insertion of the P-element in the TAF250 gene and to create stronger TAF250 alleles, the EP(3)0421 P-element was excised by introducing a source of transposase. Viable lines in which the P-element was precisely excised no longer display bristle and sterility phenotypes observed in the parental line, indicating that the P-element is the cause of these phenotypes. One of the lethal excision lines, EP(3)0421EX1, was analyzed in detail. Sequencing of the TAF250 gene from EP(3)0421EX1 reveals that the parental P-element was internally deleted, leaving behind 52 bp of P-element sequence. The small insertion contains an in-frame stop codon within the first exon of TAF250, and therefore may be a null allele. In fact, like Df(3R)BD5, which deletes the TAF250 gene, EP(3)0421EX1 fails to complement the lethality of all SR3-5 alleles. EP(3)0421 and SR3-5 mutants have been renamed TAF250 (Wassarman, 2000).

Trans-heterozygous combinations of lethal TAF250 alleles and a viable P-element allele TAF250EP421 exhibit developmental phenotypes. The severity and nature of these defects depends on the allelic combination. All allelic combinations display defects in oogenesis, ocelli development, and developmental timing; in addition, flies with certain mutant combinations display defects in eye, wing, bristle, and terminalia development, indicating roles for TAF250 in multiple developmental pathways (Wassarman, 2000).

Mutations in TAF250 disrupt development of photosensitive organelles, the eyes and ocelli. Adult compound eyes are composed of hundreds of ommatidia that are arranged in a perfect hexagonal array with single mechanosensory bristles that project from alternate vertices of ommatidia. In TAF250XS-2232/TAF250EP421 flies, ommatidia are irregularly shaped and are not aligned in straight rows. In addition, the number of interommatidial bristles is reduced, multiple bristles sometimes originate from a single site, and bristles are randomly distributed (Wassarman, 2000).

The number of photoreceptor cells per ommatidium is altered in TAF250 adult flies. Apical tangential sections through wild-type eyes reveal a trapezoid pattern of photoreceptors, with six large outer photoreceptors (R1-6) forming the perimeter of the trapezoid and one small inner photoreceptor (R7) in the center of the trapezoid. In TAF250XS-2232/TAF250EP421 flies, approximately 10% of the ommatidia are missing one outer photoreceptor and 14% have one extra outer photoreceptor. TAF250 mutations also disrupt development of ocelli. Three ocelli are normally arranged in a triangle on the top of the head. Severe TAF250 mutants completely lack individual ocelli whereas less severe mutants have ocelli that are reduced in size (Wassarman, 2000).

Mutations in TAF250 alter bristle development on the thorax and head. The adult cuticle of D. melanogaster has numerous precisely positioned large bristles, macrochaete, and small bristles, microchaete. On the thorax, ten rows of microchaete bristles are positioned between pairs of macrochaete bristles. In TAF250 mutants, several bristles in each row are shifted slightly, disrupting the orderly pattern of bristle rows. Microchaete size and shape do not appear to be affected and all aspects of macrochaete morphology appear normal on the thorax. In contrast, the number of bristles surrounding the ocelli is reduced, including complete elimination of ocellar bristles (Wassarman, 2000).

Mutations in TAF250 result in incomplete rotation of the terminal A9 body segment in males. It has been hypothesized that during pupation, the developing terminalia undergo a 360o rotation in a clockwise direction to achieve the configuration of ejaculatory and tracheal ducts and the posterior peripheral nerves observed in adult male flies (Wassarman, 2000). Incomplete rotation is observed in approximately 5% of TAF250XS-2232/TAF250EP421 males. Different degrees of incomplete rotation occur. In the most extreme phenotype the terminalia have rotated only 30° and the last tergite and analplate protrudes from the body. It should be noted that this phenotype could be interpreted as a 30° overrotation of the terminalia (Wassarman, 2000).

Mutations in TAF250 disrupt wing development. In TAF250 flies, the fourth longitudinal vein (L4) bifurcates along the wing margin forming a delta. The shape and size of the wings appear normal as does the morphology of the other veins. However, the wings are often held out to the sides of the body, rather than crossed over the body. TAF250 mutants are also female sterile. Mutations in TAF250 do not appear to affect the fertility of males. In the most severe case, TAF250XS-2232/TAF250EP421, the ovaries are very small and the flies lay very few eggs that do not develop. In a less severe case, TAF250S-625/TAF250EP421, the ovaries appear normal in size and the flies lay more eggs but they do not develop. Examination of ovaries dissected from TAF250S-625/TAF250EP421 flies reveals that approximately 5% of egg chambers contain 8 cells (7 nurse cells and 1 oocyte) in contrast to wild-type egg chambers that contain 16 cells (15 nurse cells and 1 oocyte). Eight-cell egg chambers may result from a reduction in the number of cell divisions, from four to three, that a single cystoblast normally undergoes to generate a 16-cell egg chamber (Wassarman, 2000).

TAF250 is required for normal developmental timing. Mutations in TAF250 cause a delay in development. This was determined by examining how long transheterozygous TAF250 mutants take to develop from an egg to adult relative to their heterozygous mutant siblings. The strongest affect is observed with TAF250XS-2232/TAF250EP421 flies that are delayed approximately two days in development, but all transheterozygous TAF250 combinations are delayed to some extent. Delayed eclosion of mutant flies could reflect slowed developmental processes or an inability to gather enough nutrients.

The SR3-5 mutations identify a conserved region of TAF250 that is critical for its function. The TAF250-coding region of the SR3-5 alleles was sequenced. In each case, single amino acid changes were observed that lie within a 55-amino acid central region of TAF250. The TAF250S-136 gene product has an Asp-1041 to Asn change; TAF250XS-2232 has a Val-1072 to Asp change, and TAF250S-625 has an Arg-1096 to Pro change. These substitutions alter residues that are conserved within metazoan organisms but are not conserved in the homologous yeast protein TAF145 and thus potentially identify a TAF250 function that is specific to metazoan organisms (Wassarman, 2000).

The point mutations in TAF250 fall within a region that has been shown to interact with transcriptional regulatory proteins, such as HIV TAT and retinoblastoma protein, and to possess HAT activity. It should be noted that the TAF250 HAT domain does not contain sequence similarity to the acetyl-CoA binding site defined for acetyltransferases including GCN5. To test whether HAT activity is altered by any of the TAF250 mutations, a recombinant polypeptide encompassing amino acids 612-1,140 (TAF250-M) of Drosophila TAF250 was produced in bacteria, and site-directed mutagenesis was used to generate the identified single amino acid changes within the context of this polypeptide. Purified recombinant proteins were tested in vitro for HAT activity by an activity gel assay. This assay revealed that the TAF250-M region of TAF250 has HAT activity, which narrows down the HAT domain to a small region. Interestingly, the point mutations do not significantly affect this activity. This result indicates that the mutations do not affect the catalytic activity of the TAF250 HAT, but it does not exclude the possibility that they regulate substrate specificity or the kinetics of the acetylation reaction (Wassarman, 2000).

TAFs were originally defined biochemically as components of a complex, TFIID, that is required for activator-responsive transcription in vitro. Subsequent in vitro studies have shown that complexes containing only a subset of TAFs function similarly to holo-TFIID, and specific biochemical functions have been attributed to individual TAFs. These findings raise the question of whether all TAFs are required for the transcription of TAF-dependent genes in vivo. The isolation of mutations in TAF60, TAF110, and TAF250 in the same genetic screen suggests that some genes require the function of multiple TAFs. This is supported by the finding that (1) TAF60 and TAF110 are required for the transcription of twist and snail genes in the Drosophila embryo; (2) TAF60 and TAF250 mutants have similar adult phenotypes; and (3) the transcription of some genes in yeast depends on multiple TAFs. Conversely, because all Drosophila TAFs were not isolated in the sev-Ras1V12 screen, it suggests that not all TAFs are required for the transcription of TAF-dependent genes or that the dose sensitivities of TAFs are different (Wassarman, 2000).

TAF250 mutants were isolated as modifiers of a Ras1-induced phenotype and display phenotypes similar to those of flies mutant for components of Ras1 signaling pathways. In Drosophila, the epidermal growth factor receptor (EGFR) tyrosine kinase signals through Ras1 to control cell proliferation, survival, and differentiation. Similar to mutations in TAF250, mutations in EGFR or downstream components of EGFR signaling pathways cause female sterility, rough eyes with associated bristle defects, altered photoreceptor number, loss of ocelli and ocellar bristles, and extra wing vein material. In addition, misrotated terminalia are observed in the head involution defective (hid) mutant. hid encodes a pro-apoptotic factor that is transcriptionally regulated by the EGFR/Ras1 pathway. The correlation between TAF250 and EGFR pathway mutant phenotypes suggests that TAF250 transcriptionally regulates genes in the EGFR pathway (possibly EGFR, which is a target for TAF250 in mammalian cells, or downstream genes in the pathway, like hid). Alternatively, some of the TAF250 phenotypes may result from altered transcription of ribosomal protein genes. Minute mutants encode ribosomal proteins and have common phenotypes, including delayed development, female sterility, and short and slender bristles . Furthermore, some Minutes have rough eyes, missing ocelli, or misrotated terminalia. TAF250 mutants display all of these phenotypes, except for changes in bristle morphology, suggesting that, as in yeast, TAF250 regulates ribosomal protein gene transcription. Finally, the oogenesis defect suggests a role for TAF250 in cell cycle progression, as has been reported for yeast and mammalian cells. Mutations in cyclin E, which is necessary for the G1/S transition, also cause the formation of eight-cell egg chambers, suggesting that TAF250 may regulate transcription of cyclin E (Wassarman, 2000 and references therein).


GENE STRUCTURE

cDNA clone length - 6396

Bases in 5' UTR - 77

Bases in 3' UTR - 248


PROTEIN STRUCTURE

Amino Acids - 2068

Structural Domains

DTAFII230 (p230) can be divided into four subdomains. The small amino-terminal domain (I) (amino acids 1-235) is a highly acidic region that is moderately conserved between Drosophila and human (25% identity). This domain is also rich in glycine and serine residues, suggesting that it might form a fairly flexible and/or closely packaged structure as a result of the small side chains of these amino acids. In contrast, the large domain II ( amino acids 236-1706) is well conserved (55% identity), especially in its central region (78% identity for amino acids 610-1178). Domain II contains three intriguing structural motifs: an HMG box (amino acids 1246-1360); a potential nuclear localization signal (amino acids 1445-1452); and 130-amino-acid direct repeats containing bromodomain in each repeat (amino acids 1457-1577 and 1578-1699). Domain III (1707-1993 amino acids) is barely conserved in primary sequence (15% identity) between Drosophila and human, but both species contain a remarkable number of acidic residues (Drosophila 24%). This acidic domain has two consecutive negative charges, both of which are consensus target sites for casein kinase II, which is known to control the activity of several transcription factors. The most C-terminal domain (IV) (amino acids 1994-2068) is a glutamine-rich tail present in the Drosophila sequence, but not in the human sequence (Kokubo, 1993).

Remarkably, only the HMG box of p230 is interrupted by the insertion of a 35-amino acid sequence. Other HMG box proteins of Drosophila include HMG-D and Sox box protein 70D(also known as Dichaete). It is unknown whether the HMG box of p230 is active in DNA binding and bending. The bromodomain is a feature common to several global transcriptional activators, such as SNF2 of yeast and Brahma of Drosophila, as well as Imitation SWI and Male specific lethal 2 (Kokubo, 1993).

A kinase domain resides within the N-terminal portion of Taf1. There are several patches of amino acid sequences with significant but weak homology to the protein kinase family. The best match to multiple kinase homologies is found within the N-terminal region of both Drosophila and human Taf1. A minimal kinase domain is located in human Taf1, mapping to residues 1-434. Deletion of residues 1-161 abrogates phosphorylation of RAP74, also known as TFIIF (see RNA polymerase and general transcription factors) as well as the potential for autophosphorylation. A second distinct kinase domain is located at the C-terminal region of Taf1. An autophosphorylation domain is intrinisic to the C-terminal 468 amino acid residues of hTaf1. The amino acid sequence of this putative C-terminal kinase (CTK) shares no apparent homology to other known protein kinases (Dikstein, 1996).

Drosophila Taf1 possesses a histone transacetylase (HAT) activity that targets histone H3 and histone H4. The putative catalytic domain of Drosophila Taf1 is located between residues 1 and 1140. Furthermore, sequences between residues 885 and 1140 are critical for HAT activity. This region corresponds to a highly conserved domain shared with yeast TAFII130, the homolog of Drosophila Taf1. The central portion of yeast TAFII130 possesses the HAT activity. There is no similarity of Taf1 or yeast TAFII130 to other known acetyltransferase proteins. These results suggest that the HAT catalytic domain of Taf1 may represent a second type of HAT domain, unrelated to previously characterized histone acetyltransferases (Mizzen, 1996).


TBP-associated factor 1: Evolutionary Homologs | Regulation | References

date revised: 6 Jan 97 

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