Abdominal-B
Abdominal-B homologs interaction with Extradenticle homologs Dimerization with Extradenticle or the mammalian Exd homologs, the PBX homeoproteins, dramatically improves DNA binding by HOX
transcription factors, indicating that recognition by such complexes is important for HOX specificity. For
HOX monomeric binding, a major determinant of specificity is the flexible N-terminal arm. It makes
base-specific contacts via the minor groove, including one to the 1st position of a 5'-TNAT-3' core by a
conserved arginine (Arg-5). Arg-5 also contributes to the stability of HOX.PBX
complexes, apparently by forming the same DNA contact. Heterodimers of PBX
with HOXA1 (Drosophila homolog: labial) or HOXD4 (Drosophila homolog: Deformed) proteins have different specificities at another position recognized by the
N-terminal arm (the 2nd position in the TNAT core). Significantly, N-terminal arm residues 2 and 3,
which distinguish the binding of HOXA1 and HOXD4 monomers, play no role in the specificity of their
complexes with PBX. In addition, HOXD9 and HOXD10,(Abdominal-B homologs) which are capable of binding both TTAT and
TAAT sites as monomers, can cooperate with PBX1A only on a TTAT site. These data suggest that
some DNA contacts made by the N-terminal arm are altered by interaction with PBX (Phelan, 1997).
Recent studies show that Hox homeodomain proteins from paralog groups 1 to 10 gain
DNA binding specificity and affinity through cooperative binding with the divergent
homeodomain protein Pbx1. However, the AbdB-like Hox proteins from paralogs 11,
12, and 13 do not interact with Pbx1a, raising the possibility of different protein
partners. The Meis1 homeobox gene (Drosophila homolog: homothorax) has 44% identity to Pbx within the homeodomain
and has been identified as a common site of viral integration in myeloid leukemias arising in
BXH-2 mice. These integrations result in constitutive activation of Meis1.
The Hoxa-9 gene is frequently activated by viral integration in the same
BXH-2 leukemias, suggesting a biological synergy between these two distinct classes
of homeodomain proteins in causing malignant transformation. The
Hoxa-9 protein physically interacts with Meis1 proteins by forming heterodimeric
binding complexes on a DNA target containing a Meis1 site (TGACAG) and an
AbdB-like Hox site (TTTTACGAC). Hox proteins from the other AbdB-like paralogs (Hoxa-10, Hoxa-11, Hoxd-12, and Hoxb-13) also form DNA binding complexes with
Meis1b, while Hox proteins from other paralogs do not appear to interact with Meis1
proteins. DNA binding complexes formed by Meis1 with Hox proteins dissociate
much more slowly than DNA complexes with Meis1 alone, suggesting that Hox
proteins stabilize the interactions of Meis1 proteins with their DNA targets (Shen, 1997).
Hoxa9, Meis1 and Pbx1 encode homeodomain containing proteins implicated in leukemic transformation in both mice
and humans. Hoxa9, Meis1 and Pbx1 proteins have been shown to physically interact with each other, as Hoxa9
cooperatively binds consensus DNA sequences with Meis1 and with Pbx1, while Meis1 and Pbx1 form heterodimers in
both the presence and absence of DNA. Could Hoxa9 transform hemopoietic
cells in collaboration with either Pbx1 or Meis1? Primary bone marrow cells, retrovirally engineered to overexpress
Hoxa9 and Meis1a simultaneously, induce growth factor-dependent oligoclonal acute myeloid leukemia in 3 months
when transplanted into syngenic mice. In contrast, overexpression of Hoxa9, Meis1a or Pbx1b alone, or the
combination of Hoxa9 and Pbx1b fail to transform these cells acutely within 6 months post-transplantation. Similar
results were obtained when FDC-P1 cells, engineered to overexpress these genes, are transplanted to syngenic
recipients. Thus, these studies demonstrate a selective collaboration between a member of the Hox family and one of its
DNA-binding partners in transformation of hemopoietic cells (Kroon, 1998).
The HOX/HOM superfamily of homeodomain proteins controls cell fate and segmental embryonic patterning by a mechanism that is conserved in all metazoans. The linear arrangement of the Hox genes on the chromosome correlates with the spatial distribution of HOX protein expression along the anterior-posterior axis of the embryo. Most HOX proteins bind DNA cooperatively with members of the PBC family of TALE-type homeodomain proteins, which includes human Pbx1. Cooperative DNA binding between HOX and PBC proteins requires a residue N-terminal to the HOX homeodomain, termed the hexapeptide, which differs significantly in sequence between anterior- and posterior-regulating HOX proteins. The 1.9-Å-resolution structure of a posterior HOX protein, HoxA9, is reported to be complexed with Pbx1 and DNA; the structure reveals that the posterior Hox hexapeptide adopts an altered conformation as compared with that seen in previously determined anterior HOX/PBC structures. The additional nonspecific interactions and altered DNA conformation in this structure account for the stronger DNA-binding affinity and altered specificity observed for posterior HOX proteins when compared with anterior HOX proteins. DNA-binding studies of wild-type and mutant HoxA9 and HoxB1 show residues in the N-terminal arm of the homeodomains are critical for proper DNA sequence recognition despite lack of direct contact by these residues to the DNA bases. These results help shed light on the mechanism of transcriptional regulation by HOX proteins and show how DNA-binding proteins may use indirect contacts to determine sequence specificity (LaRonde-LeBlanc, 2003).
PBX1 belongs to the TALE-class of homeodomain protein and has a wide functional diversity during development. Indeed, PBX1 is required for haematopoiesis as well as for multiple developmental processes such as skeletal patterning and organogenesis. It has furthermore been shown that PBX1 functions as a HOX cofactor during development. More recent data suggest that PBX1 may act even more broadly by modulating the activity of non-homeodomain transcription factors. To better understand molecular mechanisms triggered by PBX1 during female genital tract development, additional PBX1 partners were sought that might be involved in this process. Using a two hybrid screen, a new PBX1 interacting protein containing several zinc finger motifs was identified that was called ZFPIP for Zinc Finger PBX1 Interacting Protein. ZFPIP is expressed in embryonic female genital tract but also in other PBX1 expression domains such as the developing head and the limb buds. ZFPIP is able to bind physically and in vivo to PBX1, and it prevents the binding of HOXA9/PBX complexes to their consensus DNA site. It is suggested that ZFPIP is a new type of PBX1 partner that could participate in PBX1 function during several developmental pathways (Laurent, 2007).
Hox-MEIS interactions of Abd-B homologs AbdB-like HOX proteins form DNA-binding complexes with the TALE superclass proteins MEIS1A and MEIS1B (see Drosophila Homothorax), and trimeric complexes have been identified in nuclear extracts that include a second TALE protein, PBX. Thus, soluble DNA-independent protein-protein complexes exist in mammals. The extent of HOX/TALE superclass interactions, protein structural requirements, and sites of in vivo cooperative interaction have not been fully explored. Hoxa13 and Hoxd13 expression has been shown to not overlap with that of Meis1-3 in the developing limb; however, coexpression occurs in the developing male and female reproductive tracts (FRTs). Both HOXA13 and HOXD13 associate with MEIS1B in mammalian and yeast cells, and HOXA13 can interact with all MEIS proteins but not more diverged TALE superclass members. In addition, the C-terminal domains (CTDs) of MEIS1A (18 amino acids) and MEIS1B (93 amino acids) are necessary for HOXA13 interaction; for MEIS1B, this domain is also sufficient. Yeast two hybrid studies reveal that MEIS proteins can interact with anterior HOX proteins, but for some, additional N-terminal MEIS sequences are required for interaction. Using deletion mutants of HOXA13 and HOXD13, evidence is provided for multiple HOX peptide domains interacting with MEIS proteins. These data suggest that HOX:MEIS interactions may extend to non-AbdB-like HOX proteins in solution and that differences may exist in the MEIS peptide domains utilized by different HOX groups. Finally, the capability of multiple HOX domains to interact with MEIS C-terminal sequences implies greater complexity of the HOX:MEIS protein-protein interactions and a larger role for variation of HOX amino-terminal sequences in specificity of function (Williams, 2005).
Abdominal-B homologs: transcriptional regulation The molecular mechanisms that regulate coordinated and colinear activation of Hox gene expression in space and time remain poorly understood. Plzf regulates the spatial expression of the AbdB HoxD gene complex of mice by binding to regulatory elements required for restricted Hox gene expression and can recruit histone deacetylases to these sites. It has been shown by scanning forced microscopy that Plzf, via homodimerization, can form DNA loops and bridge distant Plzf binding sites located within HoxD gene regulatory elements. Furthermore, Plzf physically interacts with Polycomb proteins on DNA. A model is proposed by which the balance between activating morphogenic signals and transcriptional repressors such as Plzf establishes proper Hox gene expression boundaries in the limb bud (Barna, 2002).
The PLZF (promyelocytic leukemia zinc finger) gene was identified by virtue of its involvement in chromosomal translocations associated with acute promyelocytic leukemia (APL). PLZF is a nuclear protein containing at the C terminus nine Krüppel-type zinc-finger domains, which recognize specific DNA sequences. At the N terminus, PLZF contains a BTB/POZ domain, which mediates self-association and transcriptional repression when fused to a heterologous DNA binding region. PLZF functions as a transcriptional repressor through its ability to recruit, via the BTB/POZ domain, nuclear corepressors such as SMRT, N-CoR, Sin-3, and, in turn, class I and II histone deacetylases to the transcriptional complex (Barna, 2002 and references therein).
Mice with a null mutation in Plzf have been generated that show striking patterning defects in both the limb and axial skeleton, including homeotic transformations of anterior skeletal elements in the developing limb into posterior structures. These transformations are accompanied by the anteriorization and ectopic expression of each member of the 5' AbdB HoxD gene complex in the developing hindlimb. These results imply that Plzf may act as an upstream regulator of HoxD gene expression. This study defines the role of Plzf in controlling the spatial activation of the AbdB HoxD gene complex through binding to cis elements within Hox genes and recruitment of histone deacetylases as well as Polycomb proteins, in turn favoring the transition from a euchromatic to a heterochromatic chromatin state (Barna, 2002).
During limb development, coordinated expression of several Hoxd genes is required in presumptive digits. A search for the underlying control sequences upstream from the cluster resulted in the discovery of Lunapark (Lnp: Drosophila homolog: CG8735, located at 44D4), a gene that shares limb and CNS expression specificities with both Hoxd genes and Evx2, another gene located nearby. A targeted enhancer-trap approach was used to identify a DNA segment capable of directing reporter gene expression in both digits and CNS, following Lnp, Evx2, and Hoxd-specific patterns. This DNA region shows an unusual interspecies conservation, including with its pufferfish counterpart. It contains a cluster of global enhancers capable of controlling transcription of several genes unrelated in structure or function, thus defining large regulatory domains. These domains were interrupted in the Ulnaless mutation, a balanced inversion that modifies the topography of the locus. The heuristic value of these results is discussed in term of locus specific versus gene-specific regulation (Spitz, 2003).
To study the relationship between DNA replication and transcription in vivo, Hox gene activation was studied in two vertebrate systems: the embryogenesis of Xenopus and the retinoic acid-induced differentiation of pluripotent mouse P19 cells. The first cell cycles following the mid-blastula transition in Xenopus are necessary and sufficient for HoxB activation, whereas later cell cycles are necessary for the correct expression pattern. In P19 cells, HoxB expression requires proliferation, and the entire locus is activated within one cell cycle. Using synchronous cultures, it was found that activation of HoxB genes is colinear within a single cell cycle, occurs during S phase and requires S phase. The HoxB locus replicates early, whereas replication is still required for maximal expression later in S phase. Thus, induction of HoxB genes occurs in a DNA replication-dependent manner and requires only one cell cycle. It is proposed that S-phase remodelling licenses the locus for transcriptional regulation (Fisher, 2003).
Temporal colinearity of HoxB expression in P19 cells is clear only when cells are synchronized within the cell cycle, suggesting that HoxB activation is linked to progression through the cell cycle. If HoxB genes are activated in a defined order during the cell cycle, each successive gene will be activated at the same time in all cells in synchronous cultures, which would explain why differences in timing of activation (i.e., temporal colinearity) are clearly visible (Fisher, 2003).
The colinearity of expression of the HoxB domain within a single cell cycle has a striking relationship with its sensitivity to inhibition of DNA replication. Thus, the later the genes are expressed, the more they are sensitive to inhibition of DNA synthesis. The peak of sensitivity is for paralogues 6-8, whereas the border paralogs Hoxb9 and Hoxb13 at the 5' end and Hoxb1 at the 3' end are less sensitive. All HoxB genes, other than Hoxb13 (which does not obey temporal colinearity in these cells) and Hoxb1, are activated in S phase when stimulated in G2. Hoxb1 and Hoxb13 are activated before S phase and thus not surprisingly do not require S phase for correct activation (Fisher, 2003).
G1-S progression is not sufficient for Hoxb2-Hoxb9 expression, since aphidicolin, which specifically blocks DNA polymerase function, prevents HoxB expression in G2-synchronized cells. This is not due to the requirement for RA itself to act at a particular cell-cycle stage, since a transient pulse of RA can induce neural differentiation from any phase of the cell cycle, and in these experiments the control gene Cyp26, as well as Hoxb1 and Hoxb13, are activated normally in replication-inhibited cells (Fisher, 2003).
These results are compatible with current models for temporal colinearity. Colinearity of HoxD genes in the mouse embryo appears to be due in part to a polar release from transcriptional repression by a distal enhancer. Furthermore, HoxB activation along the anterior-posterior axis in chick embryos suggests that progressive opening of the HoxB locus occurs in all regions of the neural tube but genes are only expressed if cis-acting factors are present. Thus, colinearity might be achieved by two component steps: regulated derepression of the locus by DNA replication, making it permissive to regulated expression of specific transcription factors (Fisher, 2003).
It is proposed that the gradient of sensitivity to aphidicolin observed according to the position of HoxB paralogs on the chromatin domain is related to polar competition between transcriptional repressor and activator elements, in which DNA replication strongly favours expression of repressed chromatin by creating nascent DNA upon which new chromatin is formed or by restructuring the domain during S phase. In P19 cells the HoxB locus may be in a predetermined state, since it replicates early and is mainly unmethylated and activation of the entire locus is rapid, yet DNA replication is still required to allow normal Hox gene expression. Possibly, the HoxB locus in P19 cells is organized into chromatin in such a way that passage through a single cell cycle allows relief of repression over the entire locus in much the same way that a single DNA replication derepresses chromatin to allow long-range enhancer function in mouse embryos(Fisher, 2003).
In some vertebrates, cell-cycle lengths show an anterior-posterior gradient (long to short), and there is a caudal-rostral wave of initiation of HoxB expression in the neural tube in these embryos. Furthermore, cell-cycle synchrony occurs in developing somites, which also show waves of HoxB expression. As such, the spatial and temporal organization of proliferation could provide a template for developmental gene expression in which staggered DNA replication along a developing axis relieves gene expression from transcriptional silencing (Fisher, 2003).
Abdominal-B homologs, and Trithorax and Polycomb group genes In Drosophila and mouse, Polycomb group genes are involved in the maintenance of homeotic gene
expression patterns throughout development. Skeletal phenotypes are found in mouse compound mutants
for two Polycomb group genes bmi1 (Drosophila homolog Sex combs reduced) and M33 (Drosophila homolog: Polycomb). Mice deficient for both bmi1 and M33 present
stronger homeotic transformations of the axial skeleton as compared to each single Polycomb group mutant,
indicating strong dosage interactions between those two genes. These skeletal transformations are
accompanied with an enhanced shift of the anterior limit of expression of several Hox genes in the somitic
mesoderm. These results demonstrate that in mice the Polycomb group genes act in synergy to control the
nested expression pattern of some Hox genes in somitic mesodermal tissues during development (Bel, 1998).
When Pc-G mutant mice are compared, loss of each Pc-G
gene shows a unique subset of affected Hox genes. For example, in M33 mutant mice, only anterior shifts for Hoxa3 can be detected and, in some cases, for Hoxc8. mel18 -/-, bmi1 -/- and rae28 -/- mice
present a more extensive overlap in affected Hox genes,
encompassing one prevertebrae anterior shifts of Hoxa5 and Hoxc8.
However, Hoxc6 and Hoxc5 are uniquely affected in bmi1 -/-
mice, while Hoxa7 and Hoxd4 are only affected in mel18 -/-
mice and Hoxb5 is unaffected in all those mutant mice. In M33 bmi1 double mutant mice, the
anterior limit of expression of at least two Hox genes, Hoxc9
and Hoxc8, is significantly more severely shifted as compared
to both single mutants, demonstrating the additive effect of
Pc-G products in maintaining the boundaries of selected Hox
genes. One striking observation is that deleting three gene doses
of these Pc-G genes does not increase the derepressive effect
of Hoxc8 or Hoxc9 expression; full deficiency of M33 and
bmi1 is required to induce extensive ectopic expression of
these two genes in mesodermal tissues. Nevertheless,
according to the differential expression of Hoxc8 and Hoxc9
in both single mutants, it seems that Hoxc8 is more sensitive
to M33 regulation since a one-segment anterior
shift is observed in M33 -/- bmi1 +/- (prevertebrae 10), whereas that shift is not visible
in bmi1 -/- M33 +/- (prevertebrae 11); reciprocally, Hoxc9 is more
sensitive to bmi1 regulation.
In contrast, some other Hox genes like
Hoxb1, Hoxd4 and Hoxd11 are not affected, either in single
or in double mutant mice. This suggests that several murine
multimeric Pc-G protein complexes of different composition
might exist that differ in their affinities for specific Hox
genes. Alternatively, since in mammals Pc-G genes exist as
highly related gene pairs (such as mel18/bmi1, Enx1/Enx2,
M33/MPc2, hPc1/hPc2 and Hph1/rae28/Hph2), a potential redundancy likely exists. This
suggests that the homologous gene complements part of the
function and thus maintains the boundaries of expression for
a subset of Hox genes. Analysis of double mutant mice for a
related pair such as mel18 and bmi1 should clarify the degree
of redundancy. Differential effects of Pc-G mutations on Hox
gene expression in different tissues like the somitic
mesoderm and the neural tube, also suggest that different Pc-G
complexes may regulate a specific subset of Hox genes in
a tissue-dependent manner (Bel, 1998).
The Polycomb group genes are required for the correct expression of the homeotic complex genes and segment specification
during Drosophila embryogenesis and larval development. In mouse, inactivation studies of several Polycomb group genes
indicate that they are also involved in Hox gene regulation. M33 mutants have been used to study
the function of M33, the mouse homolog of the Drosophila Polycomb gene. In the absence
of M33, the window of Hoxd4 retinoic acid (RA) responsiveness is opened earlier and Hoxd11 gene expression is
activated earlier in development This indicates that M33 antagonizes the RA pathway and has a function in the
establishment of the early temporal sequence of activation of Hox genes. Despite the early activation, A-P boundaries are
correct in later stages, indicating a separate control mechanism for early aspects of Hox regulation. This raises a number of
interesting issues with respect to the roles of both Pc-G proteins and Hox regulatory mechanisms. It is proposed that a
function of the M33 protein is to control the accessibility of retinoic acid response elements in the vicinity of Hox genes
regulatory regions by direct or indirect mechanisms or both. This could provide a means for preventing ectopic
transactivation early in development and be part of the molecular basis for temporal colinearity of Hox gene
expression (Bel-Vialar, 2000).
A model proposes that each gene within a Hox cluster would show a graded sensitivity to RA through a repression imposed by Pc-G genes: with 3' genes being accessible earlier than the adjacent 5' gene. This
differential repression contributed by M33 can be hypothesized
since, in two different backgrounds in M33-/-
mice, the skeletal transformations are found more frequently
in the anterior (due to the deregulation of more 3'
Hox genes) than in the posterior regions.
This would imply that the Pc-G repression is more dependent
on the M33 product at the 3' end of the cluster than at
the 5' end, or/and that the nature of the complex may vary
from the 3' end to the 5' end of a Hox cluster. This
repression could again be mediated by direct or indirect
mechanisms (Bel-Vialar, 2000).
MLL, the human homolog of Drosophila trithorax, maintains Hox gene expression in mammalian embryos and is rearranged in human leukemias resulting in Hox gene deregulation. How MLL or MLL fusion proteins regulate gene expression remains obscure. MLL regulates target Hox gene expression through direct binding to promoter sequences. The MLL SET domain is a histone H3 lysine 4-specific methyltransferase whose activity is stimulated with acetylated H3 peptides. This methylase activity is associated with Hox gene activation and H3 (Lys4) methylation at cis-regulatory sequences in vivo. A leukemogenic MLL fusion protein that activates Hox expression had no effect on histone methylation, suggesting a distinct mechanism for gene regulation by MLL and MLL fusion proteins (Milne, 2002).
How MLL regulates Hox gene expression is poorly understood. The domain structure of MLL is complex, making it difficult to unravel the key components of MLL function. Domains that may have a role in MLL function include the AT hooks, which bind DNA, a region homologous to DNA methyl transferases (DNMT), the cysteine-rich PHD domain, and a highly conserved SET domain. The SET domain is found in many proteins now demonstrated to mediate lysine-directed histone methylation. These findings suggest a possible role for MLL in chromatin remodeling mediated by histone methylation. However, early studies of this domain in MLL did not reveal evidence of enzymatic activity, leaving its function enigmatic. Furthermore, rearrangements of MLL that occur in leukemia consistently delete the PHD and SET domains and replace these sequences with one of over 30 different translocation partners that in general share little sequence homology (Milne, 2002).
Progress in understanding the mechanistic role of MLL in maintenance and gene regulation has also been slowed by a lack of known target binding sites for mammalian PcG or trxG homologs. To address these issues, attention was focused on how MLL regulates transcription of Hox c8. This target was chosen because it is tightly regulated by MLL and because it is the only Hox gene in which the sequences required for the correct initiation and maintenance of expression have been extensively mapped in vivo. Hox c8 is upregulated by MLL, supporting a transcriptional activating role for MLL. MLL binds directly to proximal promoter sequences but not to other regions of the Hox c8 locus, including the 5' and 3' enhancer sequences, suggesting that MLL-dependent regulatory elements in mammalian Hox genes are organized differently from those in Drosophila. The Hox c8 promoter is necessary and sufficient for MLL responsiveness and, along with the 5' enhancer, exhibits differential histone acetylation and H3 (Lys4) methylation in Mll+/+ as compared to Mll-/- cells. Reexpression of MLL in null cells results in methylation of H3 (Lys4) at the Hox c8 5' enhancer and promoter as well as at other Hox gene promoters. H3 (Lys4) methylation is dependent on an intact MLL SET domain and this methyltransferase activity is stimulated by H3 peptides that are acetylated at Lys9 or Lys14. Collectively, these experiments underscore the importance of a concerted series of histone and DNA modifications in the regulation and maintenance of target genes during mammalian development and provide a framework for comparing mechanisms of epigenetic forms of gene regulation by MLL and MLL fusion proteins (Milne, 2002).
Nakamura, T., et al. (2002). ALL-1 is a histone methyltransferase that ALL-1 is a member of the human trithorax/Polycomb gene family and is also involved in acute leukemia. ALL-1 is present within a stable, very large multiprotein supercomplex composed of ~29 proteins. The majority of the latter are components of the human transcription complexes TFIID (including TBP), SWI/SNF, NuRD, hSNF2H, and Sin3A. Other components are involved in RNA processing or in histone methylation. The complex remodels, acetylates, deacetylates, and methylates nucleosomes and/or free histones. The complex's H3-K4 methylation activity is conferred by the ALL-1 SET domain. Chromatin immunoprecipitations show that ALL-1 and other complex components examined are bound at the promoter of an active ALL-1-dependent Hox a9 gene. In parallel, H3-K4 is methylated, and histones H3 and H4 are acetylated at this promoter (Nakamura, 2002).
Strikingly, most ALL-1-associated proteins can be classified into well-known complexes involved in transcription. Of these, the SWI/SNF(BRM) and NuRD complexes and the hSNF2H protein are ATP-dependent chromatin remodelers: Sin3A and NuRD are histone deacetylases; two human homologs of components of the yeast Set1 complex (but not the Set1 protein) are involved in H3-K4 methylation, and TFIID acts in promoter recognition and in mediating activator responsiveness. The identification of TFIID components, including TBP, within the ALL-1 supercomplex is one of the most significant observations of this work. This finding indicates a direct connection between ALL-1 and the general transcription machinery. Several TFIID proteins have been identified as components of the Drosophila Polycomb multiprotein complex. Considering the known functions of the other complexes included within the ALL-1 supercomplex, SWI/SNF and hSNF2H may act both as activators and repressors, but Sin3A and NuRD complexes have been generally associated with transcriptional silencing. Since ALL-1 is an activator, the inclusion of these last two complexes within the ALL-1 supercomplex is surprising. However, HDAC1, a component of both Sin3A and NuRD complexes, has been found bound to active promoters of some Drosophila genes, including the trithorax-regulated Abd-B. Further, histone deacetylation might be required to enable H3-K4 methylation. Also, deacetylation might be applied to modulate the level of histone acetylation conferred by the ALL-1 complex and/or by acetyltransferases transiently associated with ALL-1. Moreover, the deacetylating complexes might target transcription factors regulated by acetylation. Finally, the inclusion within the ALL-1 supercomplex of CPSF and Symplekin involved in polyadenylation and of p116 associated with splicing provides support for the notion of direct connection between the promoter of a gene and how its transcript is processed (Nakamura, 2002and references therein).
A major finding in this work is that the ALL-1 SET domain methylates H3-K4. Previous attempts to show methyltransferase activity of ALL-1 SET were unsuccessful, probably due to the low activity of this domain (at least 10-fold lower than the activity of SUV39H1 SET, which methylates H3-K9). Presently, two other proteins have been implicated in H3-K4 methylation. Human Set7/Set9 possess this intrinsic enzymatic activity conferred by the SET domain. A Saccharomyces cerevisiae complex containing the Set1 protein methylates H3-K4, and mutation analysis of the gene implicates it directly in that histone modification in yeast. Whereas human SET7/SET9 activates transcription, yeast Set1 represses transcription of ribosomal DNA. Nevertheless, diverse findings correlate H3-K4 methylation with an active state of transcription. Thus, this modification is specifically associated with transcriptionally active macronuclei but not with inactive micronuclei in Tetrahymena. Also, immunofluorescence studies of human female chromosomes show that H3-K4 methylation accumulates at transcribed regions of autosomes but is largely excluded from the inactive X chromosome. Moreover, ChIP experiments at the mating type locus of fission yeast have shown that, while histone H3-K4 methylation is localized to actively transcribed regions, H3-K9 methylation is detected in silent heterochromatin. Similar results have been observed in ChIP analysis of the beta-globin locus during erythropoiesis (Nakamura, 2002 and references therein).
Polycomb group (PcG) proteins are essential for accurate axial body patterning during embryonic development. PcG-mediated repression is conserved in metazoans and is targeted in Drosophila by Polycomb response elements (PREs). However, targeting sequences in humans have not been described. While analyzing chromatin architecture in the context of human embryonic stem cell (hESC) differentiation, a 1.8kb region between HOXD11 and HOXD12 (D11.12) was deciphered that is associated with PcG proteins, becomes nuclease hypersensitive, and then shows alteration in nuclease sensitivity as hESCs differentiate. The D11.12 element repressed luciferase expression from a reporter construct and full repression required a highly conserved region and YY1 binding sites. Furthermore, repression was dependent on the PcG proteins BMI1 and EED and a YY1-interacting partner, RYBP. It is concluded that D11.12 is a Polycomb-dependent regulatory region with similarities to Drosophila PREs, indicating conservation in the mechanisms that target PcG function in mammals and flies (Woo, 2010).
The D11.12 element has several characteristics of a Drosophila
PRE, indicating that there is conservation of the mechanisms
that target PcG function. The multiple components that combine
to make a functional PRE in Drosophila are diverse and still not
fully understood. While the study of mammalian PREs is in its
infancy, there is reason to think that, like Drosophila, multiple
components might contribute to function. Roles in
D11.12 were observed for a hyperconserved region, for YY1 and the interacting
protein RYBP, and it is suggested that nucleosome free region is also central to function (Woo, 2010).
Focused was placed on D11.12 as playing a potential regulatory
role due to its depletion in nucleosome occupancy in mesenchymal stem cells,
a level of depletion that changes during differentiation. It is intriguing and somewhat counter-intuitive that sequences associated with recruiting the PcG system are nucleosome depleted. Most characterized activities of the PRC1 and
PRC2 families in vitro, including histone methylation, histone
ubiquitylation, and chromatin compaction, involve nucleosomes.
However, several studies have directly examined depletion of
nucleosomes on Drosophila PREs and their association with
PcG proteins. Dynamic accessibility of protein-binding sequences might be important for recruiting PcG complexes in vivo (Woo, 2010).
Recent studies suggest that in addition to nucleosome depletion,
high levels of histone replacement could be observed where
PcG and trxG binding sites exist. This suggests that PRE sequences in flies might be open
and dynamic, consistent also with proposals that RNA production
from these regions might be important for function. It was found that D11.12 is nuclease-sensitive and associated with the PcG proteins BMI1 and
SUZ12. Nucleosome depletion might therefore play a key role
mechanistically in establishing the ability to recruit PcG function
to a region of the genome, explaining the apparent conservation
of this feature between Drosophila and humans (Woo, 2010).
To date, there is only one known human DNA-binding protein,
YY1, which has homology to one of the Drosophila proteins
which functions to recruit PcG proteins at PREs. Several lines
of evidence suggest that YY1 is important to D11.12 function,
consistent with previous proposals based upon both functional
studies and homology to PHO. It is important to note that while YY1 appears central to D11.12 function, it is unlikely that this protein (or any protein) is generally required for mammalian PRE function. In mice, the
PRE-kr has a single YY1 binding site as determined by sequence
analysis, however this YY1 binding site is not conserved in the homologous human sequence and no other apparent YY1 binding sites are present. The contribution of the YY1 binding site at the PRE-kr was not examined. It is noted note that in reporter constructs containing D11.12, mutation
of the YY1 binding sites impacts binding of BMI1, a PRC1
component, but has little impact on binding of SUZ12, a PRC2
component. This is consistent with models in which PRC2 is recruited prior to PRC1, and suggests that different components of D11.12 might be involved differentially in recruitment of these two complexes. YY1 interacts with RYBP, which in turn interacts with three PRC1 proteins, RING1A, RING1B
and CBX2. Thus, at D11.12, YY1 might be involved primarily in
PRC1 recruitment (Woo, 2010).
A highly conserved region within D11.12, which shares
sequence homologies to organisms as evolutionarily different
as zebrafish, is essential for repressive function. This 237 bp
conserved region was required for the recruitment of both
PRC1 and PRC2 components and for full repression of the
reporter gene. In a search for potential regulatory sequences in
the Hoxd cluster, Duboule and colleagues made knockout
mice deleted of highly conserved sequences, among them the
conserved sequence in D11.12. Transgenic studies determined that deletion of
this conserved region impacted hoxd11 and hoxd12 expression,
however knockout mice with this region deleted displayed no
gross phenotype. This lack of gross phenotype might reflect
redundancy in either Hox protein function or in regulatory
elements with the entire Hoxd cluster. These previous data are
consistent with this conserved region having the potential
to contribute to regulation in mice; further analysis is needed
to determine whether there are contributions of the other
nearby elements to function of D11.12 in the genomic context.
The mouse PRE-kr element contains a conserved 450 bp
sequence within the functionally defined 3kb fragment. Comparison
of the conserved regions of D11.12 and PRE-kr using
the TRANSFAC database revealed only conserved GAGA
factor binding sites, a site defined in Drosophila that has no
known binding protein in mammals. Interestingly both conserved
region sequences were predicted to form NFRs when analyzed
by the nucleosome occupancy feature at the UCSC Genome Browser (Woo, 2010).
The D11.12 element also contains a CpG island. It has not been
tested whether this is important to D11.12 function, in part
because it is surrounded by key functional elements (namely,
the YY1 binding sites and the conserved element), making interpretation
of any deletion effect problematic. This element might
contribute to the nucleosome-free nature of D11.12, as CpG
islands in other areas have been shown to form nucleosomes
poorly thereby generating low nucleosome occupancy. It has previously been noted that there is a high correlation of PcG binding sites with CpG islands, leading to the proposal that these elements might
be a key determinant of PRE function in mammals (Woo, 2010).
The D11.12 sequence behaves as a strong activating
sequence in cells when PcG proteins are knocked down. These
knockdowns therefore change the expression from the D11.12
reporter construct by several orders of magnitude in MSCs.
A loss of association of the PcG proteins with the D11.12
construct in these cells might allow for the recruitment of activating
factors. In Drosophila there is precedent for the same
sequence being involved in repression and activation, as PRE
elements overlap with Trithorax response elements involved in
maintaining activation. It is possible that there is association of trxG components with D11.12 when PcG components have been removed (Woo, 2010).
A key aspect of PcG function is to maintain repression of
genes as cells differentiate. It is not clear to what extent PRE
sequences, as opposed to other aspects of PcG function, are
required for this heritable repression. Repression
of an integrated reporter is maintained when MSCs are differentiated
into adipocytes. In its natural location, D11.12 remains
associated with PcG proteins in adipocytes, although to a lesser
degree than in MSCs. In Drosophila, it is known that PcG association
can be plastic during differentiation and can be impacted
by local activators. A test for whether D11.12 is required for embryonic development will require that the homologous mouse sequence function in this
manner, as this type of experiment would require a genetically
tractable model system (Woo, 2010).
Hox genes are necessary for proper morphogenesis and organization of various body structures along the anterior-posterior body axis. These genes exist in clusters and their expression pattern follows spatial and temporal co-linearity with respect to their genomic organization. This colinearity is conserved during evolution and is thought to be constrained by the regulatory mechanisms that involve higher order chromatin structure. Earlier studies, primarily in Drosophila, have illustrated the role of chromatin-mediated regulatory processes, which include chromatin domain boundaries that separate the domains of distinct regulatory features. In the mouse HoxD complex, Evx2 and Hoxd13 are located ∼ 9 kb apart but have clearly distinguishable temporal and spatial expression patterns. This study reports the characterization of a chromatin domain boundary element from the Evx2-Hoxd13 region that functions in Drosophila as well as in mammalian cells. The Evx2-Hoxd13 region has sequences conserved across vertebrate species including a GA repeat motif, and the Evx2-Hoxd13 boundary activity in Drosophila is dependent on GAGA factor that binds to the GA repeat motif. These results show that Hox genes are regulated by chromatin mediated mechanisms and highlight the early origin and functional conservation of such chromatin elements (Vasanthi, 2010).
The role of chromatin organization in developmental gene regulation has been well established. In particular, chromatin organization that involves domain boundary elements has been shown to be a key feature of the regulation of homeotic genes in Drosophila . As the organization of Hox genes is well conserved among bilatarians, it is reasonable to speculate that the constraint that led to this conservation of organization is due to chromatin elements that regulate Hox genes. In general, when differentially expressed genes are in close proximity, as is often the case in Hox complexes, boundary elements are likely to be present between the genes to establish and maintain their distinct expression states. In the mouse HoxD complex, Evx2 and Hoxd13 are ∼9 kb apart and they are expressed in distinct regions in the developing embryo. This suggests the presence of a boundary within this 9 kb region that prevents the crosstalk between regulatory elements of the two flanking genes (Vasanthi, 2010).
In order to identify this putative boundary, sequence comparison of the Evx2-Hoxd13 region from different vertebrates were carried out, and a cluster of conserved sites along with a GA repeat motif was identified in all the species checked, from fish to mammals. The ∼3 kb fragment that included the GA repeats showed enhancer-blocking activity in Drosophila embryos, as well as in a human cell line, indicating the presence of a complex evolutionarily conserved boundary between Evx2 and Hoxd13 genes. The boundary activity was shown by both overlapping fragments, ED1a and ED1b, suggesting that the Evx2-Hoxd13 boundary is spread over several kilobases, unlike Drosophila boundaries that tend to be smaller, often less than 1 kb. Spread out boundary function in this region has also been suggested by an earlier study (Yamagishi, 2007). The complex nature of the Evx2-Hoxd13 boundary is also indicated by the observation that only early enhancers of ftz are effectively blocked, whereas late enhancers are able to drive expression of the lacZ reporter gene even in the presence of this boundary. This boundary activity was examined in the adult eye using a white gene enhancer and promoter interaction assay, and the results clearly showed no enhancer blocking activity in this tissue. These observations indicate that Evx2-Hoxd13 is a developmentally regulated boundary that functions in early embryos but not in late embryonic CNS and adult eye (Vasanthi, 2010).
It was also found that the boundary activity shown by the fragment containing GA-repeat motif is dependent on GAF in Drosophila. This indicates that the conserved GA sites are functionally relevant in Drosophila. Evx2 is the homolog of the even skipped (eve) gene of Drosophila, and both are thought to have evolved from a common ancestral gene Evx. In vertebrates, Evx is located near Hox clusters: Evx1 near HoxA and Evx2 near HoxD. In Drosophila, eve has moved away from the Hox cluster. The finding that a GAF-dependent boundary is present in the Evx2-Hoxd13 region is of particular interest in the light of a previous study showing that the eve gene in fly is also associated with a GAF-dependent boundary. These observations suggest that the boundary function evolved early on near the ancestral Evx gene and that the same combination has been conserved during evolution even in the organisms where the linkage between eve to Hox complex has been lost (Vasanthi, 2010).
Although several boundary-interacting factors are known in Drosophila, in vertebrates, CTCF is the only protein that has been well studied for its role in boundary function. A CTCF homolog is also present in Drosophila and is known to play a role in the Fab-8 boundary function in the BX-C. Interestingly, however, the Fab-7 boundary of the BX-C does not involve CTCF, and instead GAF plays an important role in its function and regulation. In the case of the Evx2-Hoxd13 boundary, and in agreement with earlier studies, no CTCF-binding sites are found. As in Fab-7, this boundary appears to be dependent on GAF. These observations suggest that although several factors act together to establish a boundary, some of them may be mutually exclusiv. Further studies in this direction will help in understanding the function and regulation of boundaries during development (Vasanthi, 2010).
These results strongly indicate the presence of GAGA-binding protein in vertebrates with functional similarity to that of Drosophila GAF. Earlier studies have also indicated that transcription of st-3 gene in Xenopus is regulated by GAGA sequences and GAGA factor, but the identity of vertebrate GAF has been elusive. In a separate study, c-krox/Th-POK was identified as the vertebrate homolog of GAF and was shown to binds to Evx2-Hoxd13 region in vertebrates (Matharu, 2010). These findings suggest that eve/Evx2 dependence on GAF is a feature acquired early in evolution and that even after eve separated from the Hox context, it retained this association and the functional features as seen in Drosophila. This work indicates that, in vertebrates, the ancient organization (as well as the GAF-dependent regulation) has been maintained at least at one of the Hox complexes. Finally, it is suggested that using this approach, other evolutionarily conserved cis elements and trans-acting factors involved in genomic organization and developmental gene regulation can be explored (Vasanthi, 2010).
Enhancers of Abdominal-B homologs Reporter gene analysis of the Hoxc-9 genomic region in transgenic mice allowed the identification of a positional enhancer in the
Hoxc-9 intron that drives expression in the posterior neural tube of midgestation mouse embryos in a Hoxc-9-related manner.
Sequence comparison to the chicken Choxc-9 intron reveals the existence of two highly conserved sequence elements (CSEs)
in a similar spatial arrangement. These structural similarities in the mammalian and avian lineage are mirrored by conserved
function of the chicken Choxc-9 intron in transgenic mice. Deletion analysis of the two introns suggests that full activity of
both enhancers depends on cooperation between the two CSEs located close to the respective 5' and 3' splice sites. Following
the paradigm of phylogenetically conserved developmental control mechanisms, the Hoxc-9 intragenic enhancer was tested in
Drosophila. Mouse Hoxc-9 enhancer acts in a conserved fashion in transgenic flies, conferring
posteriorly restricted reporter gene expression to the developing central nervous system in third instar larvae. This finding
indicates that the Hoxc-9 intragenic enhancer is involved in transcriptional regulatory circuits conserved between vertebrates
and arthropods (Papenbrock, 1998).
Transposition of anatomical structures along the anteroposterior axis has been a commonly used mechanism for changing body
proportions during the course of evolutionary time. Transposition in
mesodermal derivatives (vertebrae) could be attributed to transposition in the expression of Hox genes along the axial series of
somites. Transposition in the segmental arrangement of the spinal nerves can also be correlated with shifts in
the expression domains of Hox genes. Specifically, the expression domains of Hoxa-7, a-9 and a-10 in spinal
ganglia correspond in both mouse and chick to the positions of the brachial and lumbosacral plexuses, and this is
true even though the brachial plexus of chick is shifted posteriorly, relative to mouse, by seven segmental units. In spite of
these marked species differences in the boundaries of Hoxa-7 expression, cis regulatory elements located up to 5 kb upstream
of the chick Hoxa-7 gene show much functional and structural conservation with those described in the mouse. Chick Hoxa-7 and a-10 expression domains spread forward into regions of somites that are
initially negative for the expression of these genes. This is discussed as evidence that Hox expression in paraxial mesoderm
spreads forward, as earlier found for neurectoderm and lateral plate mesoderm, in a process that occurs independent of cell
movement (Gaunt, 1999).
Vertebrate Hox genes are activated in a spatiotemporal sequence that reflects their clustered organization. While this colinear relationship is a property of most
metazoans with an anterior to posterior polarity, the underlying molecular mechanisms are unknown. Previous work suggested that Hox genes were made
progressively available for transcription in the course of gastrulation, implying the existence of an element capable of initiating a repressive conformation, subsequently
relieved from the clusters sequentially. This element was sought by combining a genomic walk with successive transgene insertions upstream of the HoxD
complex followed by a series of deletions. The largest deficiency induced posterior homeotic transformations coincidental with an earlier activation of Hoxd genes.
These data suggest that a regulatory element located upstream of the complex is necessary for setting up the early pattern of Hox gene colinear activation (Kondo, 1999).
A series of transgene relocations were introduced in the posterior part of the HoxD cluster whereby Hoxd11 and Hoxd9 reporter genes were inserted between Hoxd13 and Evx2. In both cases, lacZ expression is downregulated and appears delayed in time, suggesting that cis-acting sequences carried along by the transgenes are rather inefficient when transposed posteriorly. These observations are explained as the result of a repressive mechanism acting over posterior Hox genes, possibly through a particular chromatin conformation. Insertion of reporter genes into this repressed domain would lead to their downregulation. A DNA fragment capable of organizing such a repressive domain was sought. In order to map the extent of this repression, that is, to determine at which distance from the cluster a Hoxd9 reporter gene would escape downregulation and recover its early expression profile, three relocation sites upstream of the HoxD complex were examined. It was anticipated that this distance would be rather short, due to the corresponding situation in the HoxA complex, where the distance separating Hoxa13 from Evx1, the paralogous genes of Hoxd13 and Evx2, is approximately 40 kb. Unlike Evx2, which is only 8 kb away from Hoxd13, Evx1 is expressed during gastrulation and gives an early lethal phenotype when inactivated. This suggested that colinear regulation in HoxA does not extend more than 40 kb upstream of Hoxa13. In contrast, the proximity of Evx2 to Hoxd13 would account for its colinear regulation. When inserted 16 kb upstream of Hoxd13 ('relocation point I' or
rel I), the Hoxd9 transgene behaves as if located between Evx2 and Hoxd13 and remains silent until posterior Hoxd genes are activated. By contrast, the same transgene inserted 28 kb upstream of Hoxd13 ('relocation point II' or
rel II) is activated earlier, only slightly later than when integrated randomly. Therefore, the 5' extremity of a potential repressive domain appears to lie between rel I and rel II, less than 40 kb upstream of Hoxd13. This distance corresponds to that found between Evx1 and Hoxa13, supporting a view whereby early colinear activation in all vertebrate complexes relies on the same process, a mechanism already at work before large-scale complex duplications occurred in an ancestral chordate (Kondo, 1999).
At midgestation, del II fetuses (del II contains a deletion upstream of rel II)
does not maintain expression of genes activated prematurely. For example, while Hoxd10 is expressed around the presumptive cervical region in day 7.5 embryos, expression by day 11 is restricted to the lower lumbar area, as for wild-type Hoxd10. Therefore, the break in spatial colinearity is subsequently corrected. In Drosophila, homeotic genes are regulated through two distinct phases: after an activation mostly controlled by segmentation genes, expression is refined using auto- and cross-regulatory interactions and memorized through the activity of Polycomb (Pc) and trithorax (trx) group genes. In vertebrates, the function of Pc- and trx-related genes may be required for proper expression maintenance as well. They may also be involved in early Hox gene repression and thus play a role in the activation phase. In del II mice, the absence of cross-regulatory interactions in the ectopic expression domains may lead to their disappearance, whereas expression is maintained in those domains where posterior genes are normally transcribed, hence the wild-type aspect of late expression profiles. Strong phenotypes are nevertheless observed, thus confirming that Hox genes exert important patterning functions at an early step of mesoderm development. In this context, maintenance may be required for the persistence of expression boundaries to ensure proper development of those structures that will subsequently derive from particular body levels. Such a maintenance phase, however, may not be necessary for the original colinear expression along the trunk axis (Kondo, 1999).
Four Hoxd genes (from Hoxd10 to Hoxd13) as well as Evx2 are expressed in the same presumptive digit domain, suggesting that they all respond to a single 'digit enhancer' located upstream of the cluster, an element that may concomitantly control expression in the tip of the genital bud. The walk and delete approach described above does not reveal the position of this element because for each insertion or deletion, the Hoxd9/lacZ reporter transgene shows expression in developing digit and genitalia. Therefore, it is believe that this enhancer element is located at least 30 kb upstream of Hoxd13 and can exert its control over at least 60 kb. Likewise, the brain enhancer acting on Evx2 is not deleted in del II animals. Such results illustrate the versatility of Hox gene promoters, for the same Hoxd9 sequences are able to integrate various regulatory signals at times and places (digits, brain) where Hoxd9 is normally not expressed (Kondo, 1999).
Hox genes regulate axial regional specification during animal embryonic development and are all grouped into four clusters.
The mouse HoxB cluster contains 10 genes, Hoxb1 to Hoxb9 and Hoxb13, which are all transcribed in the same direction. A mouse strain has been generated with a targeted 90-kb deletion within the HoxB cluster from Hoxb1 to Hoxb9. Surprisingly, heterozygous mice show no detectable abnormalities. Homozygous mutant embryos survive to term and exhibit an ordered
series of one-segment anterior homeotic transformations along the cervical and thoracic vertebral column and defects in sternum morphogenesis. Neurofilament staining indicates abnormalities in the IXth cranial nerve. Notably, simultaneous
deletion of Hoxb1 to Hoxb9 results in the sum of phenotypes of single HoxB gene mutants. Although a higher penetrance
is observed, no synergistic or new phenotypes are observed, except for the loss of ventral curvature at the cervicothoracic
boundary of the vertebral column. Although Hoxb13, the most 5' gene, is separated from the rest by 70 kb, it has been
suggested that it is expressed with temporal and spatial colinearity. The expression pattern of Hoxb13 is not
affected by the targeted deletion of the other 9 genes. Thus, Hoxb13 expression seems to be independent of the deleted
region, suggesting that its expression pattern could be achieved independent of the colinear pattern of the cluster or by a
regulatory element located 5' of Hoxb9 (Medina-Martinez, 2000).
Transcriptional targets of Abdominal-B homologs The myeolomonocytic cell line U937 differentiates into macrophages in response to a variety of agents. Several genes including
the cyclin-dependent kinase inhibitor p21waf1/cip1 and the homeobox gene transcription factor HOXA10 are induced at the
onset of differentiation. Ectopic expression of either gene results in U937 differentiation. In this paper, a mechanism is described
by which p21 and HOXA10 may act in concert, where HOXA10 can bind directly to the p21 promoter and, together with its
trimeric partners PBX1 and MEIS1, activate p21 transcription, resulting in cell cycle arrest and differentiation. These
experiments for the first time identify p21 as a selective target for a HOX protein and link the differentiative properties of a transcription factor and a cell cycle
inhibitor. Interestingly, various
studies indicate that both p21 and HOXA10 have opposing effects on myeloid cells at different stages. In the earliest progenitors, p21 slows growth in order
to maintain the stem cell pool, whereas a proliferative effect has been described for more mature myeloid progenitors. In U937 cells, p21 expression leads to G1 arrest and differentiation. HOXA10 expression is also associated with cell growth and some leukemias and has an antidifferentiative effect in early progenitors, whereas a differentiative and anticycling effect has been demonstrated in the intermediate-staged U937 cells. It is tempting
to conclude that the various roles of p21 at different stages of myeloid differentiation account for the apparent paradoxical effects of HOXA10. It is becoming
clear, however, that HOX proteins have their own mechanism for generating specificity, by making use of heterodimers and trimers and varying composite
cis-acting elements for function. How HOXA10 exerts its various cellular effects will only be understood when its other stage- and tissue-specific target genes
are identified (Bromleigh, 2000).
Osteoprotegerin (OPG), an osteoblast-secreted decoy receptor, specifically binds
to osteoclast differentiation factor and inhibits osteoclast maturation. Members
of the TGF-beta superfamily including BMPs stimulate OPG mRNA expression. In
this study the transcription mechanism of BMP-induced OPG
gene expression has been characterized. Transfection of Smad1 and a constitutively active BMP type IA receptor ALK3 (Q233) stimulates OPG promoter. Deletion analysis of OPG promoter has identified two Hoxc-8 binding sites that respond to BMP stimulation. GST-Hoxc-8 protein binds to these two Hox sites specifically. Consistent with the transfection results of the native promoter, ALK3 or Smad1 linker region (which interacts with Hoxc-8) stimulates the activation of the reporter construct with the two Hox sites. Overexpression of Hoxc-8 inhibits the induced promoter activity. When the two Hox binding sites are mutated, ALK3 or Smad1 linker region no longer activate the transcription. Importantly, Smad1 linker region
induces both OPG promoter activity and endogenous OPG protein expression in 2T3
osteoblastic cells. The medium from cells transfected with Smad1 linker region
expression plasmid effectively inhibits osteoclastogenesis. Collectively, these
data indicate that Hox sites mediate both OPG promoter construct activity and
endogenous OPG gene expression in response to BMP stimulation (Wan, 2001).
Members of the transforming growth factor superfamily are known to transduce
signals via the activation of Smad proteins. Ligand binding to transmembrane
cell surface receptors triggers the phosphorylation of pathway-specific Smads.
These Smads then complex with Smad 4 and are translocated to the nucleus where
they effect gene transcription. Smads 1 and 4
mediate BMP activation of the OPN promoter by inhibiting the interaction of
Hoxc-8 protein with a Hox-binding element. While specific DNA sequences are recognized by Smad complexes in several
promoters, the role of Smad-binding elements (SBEs) in activation of the OPN
promoter by members of the TGFbeta superfamily has not been previously evaluated. In this study the hypothesis was tested that a putative Smad-binding
region containing the sequence AGACTGTCTGGAC is involved in the activation of
the OPN promoter by members of the TGFbeta superfamily. Functional analyses
demonstrate that both the HBE- and Smad-binding regions are involved in
BMP-2-induced activation of the promoter, whereas, the HBE appears to be the
primary region involved in activation by TGFbeta. Deletion of the first 9 bases
in the Smad-binding region substantially reduces BMP-2-mediated activation of
the promoter. These results strongly suggest that both the Hox- and the
Smad-binding regions play a role in BMP-2-induced activation of the OPN
promoter (Hullinger, 2001).
To better define Abd-B type homeodomain function, to test
models that predict functional equivalence of all Hox genes
and to initiate a search for the downstream targets of
Hoxa13, a homeobox swap was performed by replacing
the homeobox of the Hoxa11 gene with that of the Hoxa13
gene. The Hoxa11 and Hoxa13 genes are contiguous Abd-B
type genes located at the 5' end of the HoxA cluster. The
modified Hoxa11 allele (A1113hd) shows near wild-type
function in the development of the kidneys, axial skeleton
and male reproductive tract, consistent with functional
equivalence models. In the limbs and female reproductive
tract, however, the A1113hd allele appears to assume
dominant Hoxa13 function. The uterus, in particular,
shows a striking homeotic transformation towards cervix/vagina, where Hoxa13 is normally expressed. Gene chips were used to create a molecular portrait of this tissue conversion and reveal over 100 diagnostic gene
expression changes. This work identifies candidate
downstream targets of the Hoxa13 gene and demonstrates
that even contiguous Abd-B homeoboxes have functional specificity (Zhao, 2001).
Mammalian Hox genes encode transcription factors that are crucial for proper
morphogenesis along the various body axes. Despite their extensive structural
and functional characterization, the nature of their target genes remains
elusive. This question was addressed by using DNA microarrays to screen for
genes whose expression in developing distal forelimbs and genital eminences is
significantly modified in the absence of the full Hoxd gene complement. This
comparative approach not only identified specific candidate genes, but also
allowed the examination of whether a similar Hox expression pattern in distinct
tissues leads to the modulation of the same or different downstream genes. This study reports
a set of potential target genes, most of which were not previously
known to play a role in the early stages of either limb or genital bud
development. Interestingly, it was found that the majority of these candidate genes
are differentially expressed in both structures, although often at different
times. This supports the idea that both appendices involve similar genetic
controls, both upstream and downstream of the Hox gene family. These results
highlight the surprising mechanistic relationship between these rather
different body parts and suggest a common developmental strategy to build up
the most distal appendicular structures of the body, i.e., the digits and the
penis/clitoris (Cobb, 2005).
Fourteen genes were identified and validated as potential HOXD targets. Six of
these (Hoxa11, Sgk, Gfra2, Epha3, Odz4 and Gdf10) are
especially strong candidates. Interestingly, with the exception of
Hoxa11, these genes are similarly regulated in limbs and genitals,
which further indicates that these two structures display closely related
developmental strategies. Initially, these genes were not found to be
regulated simultaneously in both developing buds, until the analyses were
extended to various time-points. This revealed that the same variations in
gene regulation were observed in both structures, but usually slightly later
in genitalia than in limb buds (Gfra2 is an exception to this trend).
This observation nicely fits the developmental delay that exists
between these buds, because the genital eminence emerges with a one to two day
delay with respect to forelimb budding (Cobb, 2005).
Strikingly, except for Hoxa11, the function of the five other
potential target genes has not yet been fully explored in limb or genital
development. In fact, none of the 14 candidate genes are members of the
classical FGF, BMP, WNT or SHH signaling pathways. Only the retinoid pathway
(Aldh1a2, Stra6) is represented among the candidates. This
observation supports the conclusion that, at the developmental stages
analyzed, the Hoxd genes act downstream or independently of most of the
previously described limb and genital patterning pathways (Cobb, 2005).
Geminin inhibits Hox function Embryonic development is tightly controlled. The clustered genes of the Hox family of homeobox proteins play an important part in regulating this development and also proliferation. They specify embryonic structures along the body axis, and are associated with normal and malignant cell growth. The cell-cycle regulator geminin (see Drosophila Geminin) controls replication by binding to the licensing factor Cdt1, and is involved in neural differentiation. Murine geminin associates transiently with members of the Hox-repressing polycomb complex, with the chromatin of Hox regulatory DNA elements and with Hox proteins. Gain- and loss-of-function experiments in the chick neural tube demonstrate that geminin modulates the anterior boundary of Hoxb9 transcription, which suggests a polycomb-like activity for geminin. The interaction between geminin and Hox proteins prevents Hox proteins from binding to DNA, inhibits Hox-dependent transcriptional activation of reporter and endogenous downstream target genes, and displaces Cdt1 from its complex with geminin. By establishing competitive regulation, geminin functions as a coordinator of developmental and proliferative control (Luo, 2004).
Degradation of Abdominal-B homologs Hox proteins are transcription factors involved in controlling axial patterning, leukemias and hereditary malformations. HOXC10 oscillates in abundance during the cell cycle, being targeted for degradation early in mitosis by the ubiquitin-dependent proteasome pathway. Among abdominal-B subfamily members, the mitotic proteolysis of HOXC10 appears unique, since the levels of the paralogous HOXD10 and the related homeoprotein HOXC13 are constant throughout the cell cycle. When two destruction box motifs (D-box) are mutated, HOXC10 is stabilized and cells accumulate in metaphase. HOXC10 appears to be a new prometaphase target of the anaphase-promoting complex (APC), since its degradation coincides with cyclin A destruction and is suppressed by expression of a dominant-negative form of UbcH10, an APC-associated ubiquitin-conjugating enzyme. Moreover, HOXC10 co-immunoprecipitates the APC subunit CDC27 (see DrosophilaCdc27); its in vitro degradation is reduced in APC-depleted extracts or by competition with the APC substrate cyclin A. These data imply that HOXC10 is a homeoprotein with the potential to influence mitotic progression, and might provide a link between developmental regulation and cell cycle control (Gabellini, 2003).
Noncoding RNAs (ncRNA) participate in epigenetic regulation but are poorly understood. This study characterized the transcriptional landscape of the four human HOX loci at five base pair resolution in eleven anatomic sites, and identified 231 HOX ncRNAs that extend known transcribed regions by more than 30 kilobases. HOX ncRNAs are spatially expressed along developmental axes, possess unique sequence motifs, and their expression demarcate broad chromosomal domains of differential histone methylation and RNA polymerase accessibility. A 2.2 kilobase ncRNA was identified residing in the HOXC locus, termed HOTAIR, which represses transcription in trans across 40 kilobases of the HOXD locus. HOTAIR interacts with Polycomb Repressive Complex 2 (PRC2) and is required for PRC2 occupancy and histone H3 lysine-27 trimethylation of HOXD locus. Thus, transcription of ncRNA may demarcate chromosomal domains of gene silencing at a distance; these results have broad implications for gene regulation in development and disease states (Rinn, 2007).
By analyzing the transcriptional and epigenetic landscape of the HOX loci at high resolution in cells with many distinct positional identities, a panoramic view was obtained of multiple layers of regulation involved in maintenance of site-specific gene expression. The HOX loci are demarcated by broad chromosomal domains of transcriptional accessibility, marked by extensive occupancy of RNA polymerase II and H3K4 dimethylation and, in a mutually exclusive fashion, by occupancy of PRC2 and H3K27me3. The active, PolII-occupied chromosomal domains are further punctuated by discrete regions of transcription of protein-coding HOX genes and a large number of long ncRNAs. These results confirm the existence of broad chromosomal domains of histone modifications and occupancy of HMTases over the Hox loci, and extend on those observation in several important ways (Rinn, 2007).
First, by comparing the epigenetic landscape of cells with distinct positional identities, it was showm that the broad chromatin domains can be programmed with precisely the same boundary but with diametrically opposite histone modifications and consequences on gene expression. The data thus functionally pinpoint the locations of chromatin boundary elements in the HOX loci, the existence of some of which have been predicted by genetic experiments. One such boundary element appears to reside between HOXA7 and HOXA9. This genomic location is also the switching point in the expression of HOXA genes between anatomically proximal versus distal patterns and is the boundary of different ancestral origins of HOX genes, raising the possibility that boundary elements are features demarcating the ends of ancient transcribed regions. Second, the ability to monitor 11 different HOX transcriptomes in the context of the same cell type conferred the unique ability to characterize changes in ncRNA regulation that reflect their position in the human body. This unbiased analysis identified more than 30 kb of new transcriptional activity, revealed ncRNAs conserved in evolution, mapped their anatomic patterns of expression, and uncovered enriched ncRNA sequence motifs correlated with their expression pattern -- insights which could not be gleamed from examination of EST sequences alone. The finding of a long ncRNA that acts in trans to repress HOX genes in a distant locus is mainly due to the ability afforded by the tiling array to comprehensively examine the consequence of any perturbation over all HOX loci. The expansion of a handful of Hox-encoded ncRNAs in Drosophila to hundreds of ncRNAs in human HOX loci suggests increasingly important and diverse roles for these regulatory RNAs (Rinn, 2007).
An important limitation of the tiling array approach is that while improved identification of transcribed regions is obtained, the data does not address the connectivity of these regions. The precise start, end, patterns of splicing, and regions of double-stranded overlap between ncRNAs will need to be addressed by detailed molecular studies in the future (Rinn, 2007).
The results uncovered a new mechanism whereby transcription of ncRNA dictates transcriptional silencing of a distant chromosomal domain. The four HOX loci demonstrate complex cross regulation and compensation during development. For instance, deletion of the entire HOXC locus exhibits a milder phenotype than deletion of individual HOXC genes, suggesting that there is negative feedback within the locus. Multiple 5' HOX genes, including HOXC genes, are expressed in developing limbs, and deletion of multiple HOXA and HOXD genes are required to unveil limb patterning defects. The results suggest that deletion of the 5' HOXC locus, which encompass HOTAIR, may lead to transcriptional induction of the homologous 5' HOXD genes, thereby restoring the total dosage of HOX transcription factors. How HOX ncRNAs may contribute to cross-regulation among HOX genes should be addressed in future studies (Rinn, 2007).
HOTAIR ncRNA is involved in Polycomb Repressive Complex 2-mediated silencing of chromatin. Because many HMTase complexes lack DNA binding domains but possess RNA binding motifs, it has been postulated that ncRNAs may guide specific histone modification activities to discrete chromatin loci. This study has shown that HOTAIR ncRNA binds PRC2 and is required for robust H3K27 trimethylation and transcriptional silencing of the HOXD locus. HOTAIR may therefore be one of the long sought after RNAs that interface the Polycomb complex with target chromatin. A potentially attractive model of epigenetic control is the programming of active or silencing histone modifications by specific noncoding RNAs. Just as transcription of certain ncRNA can facilitate H3K4 methylation and activate transcription of the downstream Hox genes (Sanchez-Elsner, 2006; Schmitt, 2005), distant transcription of other ncRNAs may target the H3K27 HMTase PRC2 to specific genomic sites, leading to silencing of transcription and establishment of facultative heterochromatin. In this view, extensive transcription of ncRNAs is both functionally involved in the demarcation of active and silent domains of chromatin as well as being a consequence of such chromatin domains (Rinn, 2007).
Several lines of evidence suggest that HOTAIR functions as a bona fide long ncRNA to mediate transcriptional silencing. First, full length HOTAIR is detected in vivo and in primary cells, but not small RNAs derived from HOTAIR indicative of miRNA or siRNA production. Second, depletion of full length HOTAIR led to loss of HOXD silencing and H3K27 trimethyation by PRC2, and third, endogenous or in vitro transcribed full length HOTAIR ncRNA physically associated with PRC2. While these results do not rule out the possibility that RNA interference pathways may be subsequently involved in PcG function, they support the notion that the long ncRNA form of HOTAIR is functional. The role of HOTAIR is reminiscent of XIST, another long ncRNA shown to be involved in transcriptional silencing of the inactive X chromosome. An important difference between HOTAIR and XIST is the strictly cis-acting nature of XIST. HOTAIR is the first example of a long ncRNA that can act in trans to regulate a chromatin domain. While a trans repressive role for HOTAIR was observed, the data do not permit ruling out a cis-repressive role in the HOXC locus. siRNA-mediated depletion of HOTAIR was substantial but incomplete; further, the proximity between the site of HOTAIR transcription and the neighboring HOXC locus may ensure significant exposure to HOTAIR even if the total pool of HOTAIR in the cell were depleted. The precise location of HOTAIR at the boundary of a silent chromatin domain in the HOXC locus makes a cis-repressive role a tantalizing possibility. Judicious gene targeting of HOTAIR may be required to address its role in cis-regulation of chromatin (Rinn, 2007).
The discovery of a long ncRNA that can mediate epigenetic silencing of a chromosomal domain in trans has several important implications. First, ncRNA guidance of PRC2-mediated epigenetic silencing may operate more globally than just in the HOX loci, and it is possible that other ncRNAs may interact with chromatin modification enzymes to regulate gene expression in trans. Second, PcG proteins are important for stem cell pluripotency and cancer development; these PcG activities may also be guided by stem cell or cancer-specific ncRNAs. Third, Suz12 contains a zinc finger domain, a structural motif that can bind RNA, and EZH2 and EED both have in vitro RNA binding activity. The interaction between HOTAIR and PRC2 may also be indirect and mediated by additional factors. Detailed studies of HOTAIR and PRC2 subunits are required to elucidate the structural features that establish the PRC2 interaction with HOTAIR. As is illustrated in this study, high throughput approaches for the discovery and characterization of ncRNAs may aid in dissecting the functional roles of ncRNAs in these diverse and important biological processes (Rinn, 2007).
Acute myeloid leukemia (AML) underlies the uncontrolled accumulation of
immature myeloid blasts. Several cytogenetic abnormalities have been
associated with AML. Among these is the NUP98-HOXA9 (NA9) translocation
that fuses the Phe-Gly repeats of nucleoporin NUP98 (see Drosophila Nup98-96) to the homeodomain of the transcription factor HOXA9 (see Drosophila Abd-B). The mechanisms enabling NA9-induced leukemia are poorly understood. A genetic screen in Drosophila was conducted for modifiers of NA9. The screen uncovered 29 complementation groups, including genes with
mammalian homologs known to impinge on NA9 activity. Markedly, the
modifiers encompassed a diversity of functional categories, suggesting
that NA9 perturbs multiple intracellular events. Unexpectedly, this
study discovered that NA9 promotes cell fate transdetermination and that
this phenomenon is greatly influenced by NA9 modifiers involved in
epigenetic regulation. Together, this work reveals a network of genes
functionally connected to NA9 that not only provides insights into its
mechanism of action, but also represents potential therapeutic targets (Gavory, 2021).
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