ATP-dependent chromatin remodeling by the CHD family of proteins plays an important role in the regulation of gene transcription. Here we report that full-length CHD8 interacts directly with -catenin and that CHD8 is also recruited specifically to the promoter regions of several -catenin-responsive genes. Our results indicate that CHD8 negatively regulates -catenin-targeted gene expression, since short hairpin RNA against CHD8 results in the activation of several -catenin target genes. This regulation is also conserved through evolution; RNA interference against kismet, the apparent Drosophila ortholog of CHD8, results in a similar activation of -catenin target genes. We also report the first demonstration of chromatin remodeling activity for a member of the CHD6-9 family of proteins, suggesting that CHD8 functions in transcription through the ATP-dependent modulation of chromatin structure.The alteration of chromatin structure provides a key regulatory step for all processes that act upon DNA (41). The factors that regulate this structure, commonly referred to as chromatin remodeling enzymes, can be grouped into two broad categories: complexes that alter chromatin structure via the covalent modification of histones (25,42,83) and complexes that use the energy of ATP hydrolysis to alter the structure or position of the nucleosome (6,47,57,67).ATP-dependent chromatin remodeling enzymes modulate the contacts between histones and DNA. In vitro, these enzymes catalyze structural changes that allow factors to access nucleosomal DNA, reposition nucleosomes on a template, transfer histone octamers to donor DNA, and replace histones with histone variants (27,43,80). In vivo, these activities are crucial for transcription, replication, repair, and recombination of the eukaryotic genome (2,21,59,65). These remodeling enzymes can be divided into numerous families based on domain architecture. One such family is the CHD (chromodomain, helicase, DNA binding) group of proteins, which are critical regulators of chromatin structure (23,26,29,49). These enzymes are characterized by tandem chromodomains N-terminal to their catalytic Snf2 helicase domain.The CHD family can be further subdivided into three subfamilies: CHD1-2, CHD3-5, and CHD6-9. While the first two subfamilies have been extensively studied, very little is known about the CHD6-9 family (29, 49). Previous studies have indicated that CHD8 may regulate the Wnt signaling pathway, since an N-terminal fragment of CHD8 was previously identified as a protein in Rattus norvegicus that binds -catenin both in vivo and in vitro (61). This N-terminal fragment, termed Duplin, contains only the chromodomains and lacks the Snf2 helicase domain and C-terminal sequences. Overexpression of this N-terminal fragment results in inhibition of Tcf4-dependent transcription, and studies of Xenopus embryos demonstrated that this fragment inhibited axis formation and -catenin-mediated axis duplication (61).The "canonical" Wnt signaling pathway functions by controlling the soluble pool ...
A major role of the RNAi pathway in Schizosaccharomyces pombe is to nucleate heterochromatin, but it remains unclear whether this mechanism is conserved. To address this question in Drosophila, we performed genomewide localization of Argonaute2 (AGO2) by chromatin immunoprecipitation (ChIP)-seq in two different embryonic cell lines and found that AGO2 localizes to euchromatin but not heterochromatin. This localization pattern is further supported by immunofluorescence staining of polytene chromosomes and cell lines, and these studies also indicate that a substantial fraction of AGO2 resides in the nucleus. Intriguingly, AGO2 colocalizes extensively with CTCF/CP190 chromatin insulators but not with genomic regions corresponding to endogenous siRNA production. Moreover, AGO2, but not its catalytic activity or Dicer-2, is required for CTCF/CP190-dependent Fab-8 insulator function. AGO2 interacts physically with CTCF and CP190, and depletion of either CTCF or CP190 results in genome-wide loss of AGO2 chromatin association. Finally, mutation of CTCF, CP190, or AGO2 leads to reduction of chromosomal looping interactions, thereby altering gene expression. We propose that RNAi-independent recruitment of AGO2 to chromatin by insulator proteins promotes the definition of transcriptional domains throughout the genome.
Heterochromatin protein 1 (HP1), first discovered in Drosophila melanogaster, is a highly conserved chromosomal protein implicated in both heterochromatin formation and gene silencing. We report here characterization of an HP1-interacting protein, heterochromatin protein 2 (HP2), which codistributes with HP1 in the pericentric heterochromatin. HP2 is a large protein with two major isoforms of approximately 356 and 176 kDa. The smaller isoform is produced from an alternative splicing pattern in which two exons are skipped. Both isoforms contain the domain that interacts with HP1; the larger isoform contains two AT-hook motifs. Mutations recovered in HP2 act as dominant suppressors of position effect variegation, confirming a role in heterochromatin spreading and gene silencing.
Handgrip strength is decreased in the RASopathies. The etiology of the reduced muscle force is unknown, but likely multifactorial.
a b s t r a c tChromodomain, helicase, DNA-binding protein 8 (CHD8) is an ATP-dependent chromatin remodeling enzyme that has been demonstrated to exist within a large protein complex which includes WDR5, Ash2L, and RbBP5, members of the Mixed Lineage Leukemia (MLL) histone modifying complexes. Here we show that CHD8 relocalizes to the promoter of the MLL regulated gene HOXA2 upon gene activation. Depletion of CHD8 enhances HOXA2 expression under activating conditions. Furthermore, depletion of CHD8 results in a loss of the WDR5/Ash2L/RbBP5 subcomplex, and consequently H3K4 trimethylation, at the HOXA2 promoter. These studies suggest that CHD8 alters HOXA2 gene expression and regulates the recruitment of chromatin modifying enzymes. Structured summary:MINT-7542810: CHD8 (uniprotkb:Q9HCK8) physically interacts (MI:0915) with RbBP5 (uniprotkb:Q15291) by anti tag coimmunoprecipitation (MI:0007) MINT-7542794: CHD8 (uniprotkb:Q9HCK8) physically interacts (MI:0915) with WDR5 (uniprotkb:P61964) by anti tag coimmunoprecipitation (MI:0007) MINT-7542820: CHD8 (uniprotkb:Q9HCK8) physically interacts (MI:0915) with ASH2L (uniprotkb:Q9UBL3) by anti tag coimmunoprecipitation (MI:0007) MINT-7542769: CHD8 (uniprotkb:Q9HCK8) physically interacts (MI:0914) with RbBP5 (uniprotkb:Q15291), ASH2L (uniprotkb:Q9UBL3) and WDR5 (uniprotkb:P61964) by anti tag coimmunoprecipitation (MI:0007)
RASopathies are a class of genetic syndromes caused by germline mutations in genes encoding Ras/MAPK pathway components. Cardio-facio-cutaneous (CFC) syndrome is a RASopathy characterized by distinctive craniofacial features, skin and hair abnormalities, and congenital heart defects caused by activating mutations of BRAF, MEK1, MEK2, and KRAS. We define the phenotype of seven patients with de novo deletions of chromosome 19p13.3 including MEK2; they present with a distinct phenotype but have overlapping features with CFC syndrome. Phenotypic features of all seven patients include tall forehead, thick nasal tip, underdeveloped cheekbones, long midface, sinuous upper vermilion border, tall chin, angular jaw, and facial asymmetry. Patients also have developmental delay, hypotonia, heart abnormalities, failure to thrive, obstructive sleep apnea, GE reflux and integument abnormalities. Analysis of EGF stimulated fibroblasts revealed that P-MEK1/2 was ~50% less abundant in cells carrying the MEK2 deletion compared to the control. Significant differences in total MEK2 and Sprouty1 abundance were also observed. Our cohort of seven individuals with MEK2 deletions has overlapping features associated with RASopathies. This is the first report suggesting that, in addition to activating mutations, MEK2 haploinsufficiency can lead to dysregulation of the MAPK pathway.
Drosophila melanogaster heterochromatin protein 2 (HP2) interacts with heterochromatin protein 1 (HP1). In polytene chromosomes, HP2 and HP1 colocalize at the chromocenter, telomeres, and the small fourth chromosome. We show here that HP2 is present in the arms as well as the centromeric regions of mitotic chromosomes. We also demonstrate that Su(var)2-HP2 exhibits a dosage-dependent modification of variegation of a yellow reporter transgene, indicating a structural role in heterochromatin formation. We have isolated and characterized 14 new mutations in the Su(var)2-HP2 gene. Using w m4h , many (but not all) mutant alleles show dominant Su(var) activity. Su(var)2-HP2 mutant larvae show a wide variety of mitotic abnormalities, but not the telomere fusion seen in larvae deficient for HP1. The Su(var)2-HP2 gene codes for two isoforms: HP2-L (365 kDa) and HP2-S (175 kDa), lacking exons 5 and 6. In general, mutations that affect only the larger isoform result in more pronounced defects than do mutations common to both isoforms. This suggests that an imbalance between large and small isoforms is particularly deleterious. These results indicate a role for HP2 in the structural organization of chromosomes and in heterochromatin-induced gene silencing and show that the larger isoform plays a critical role in these processes. T HE DNA found inside a eukaryotic nucleus does not exist as such, but is packaged with proteins to form chromatin. By weight, chromatin is approximately one-third DNA, one-third histones, and one-third nonhistone chromosomal proteins, plus a small RNA component. The histones play a major role in packaging and organizing the very long DNA molecules of each chromosome. Analysis of post-transcriptional modifications of histones shows that these basic proteins also play a significant role in determining specific modes of packaging and concomitant gene regulation, interacting with both enzymes and structural proteins that define alternative chromatin states (for reviews, see Richards and Elgin 2002;Khorasanizadeh 2004). One level of packaging, evident by cytological examination of interphase nuclei, is the partitioning of chromatin into euchromatin and heterochromatin. While euchromatin decondenses during interphase, heterochromatin remains relatively more condensed, showing intense staining.In Drosophila melanogaster, the entire Y chromosome, most of the fourth chromosome, the proximal 40% of the X chromosome, and the pericentric 20% of the major autosomes are heterochromatic. Euchromatic DNA has a high proportion of genes and unique sequences and tends to replicate throughout S phase. Heterochromatin has relatively few genes, is rich in repetitive sequences, and tends to replicate late in S phase; it is often found to be underreplicated (compared to euchromatin) in the polytene chromosomes of insects such as D. melanogaster. Heterochromatin is also characterized by a very low rate of meiotic recombination (Weiler and Wakimoto 1995;Zhimulev et al. 2004).Euchromatin and heterochromatin appear t...
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