Chromatin Remodeler Chd1 Research Articles

Finish the unfinished: Chd1 resolving hexasome-nucleosome complex with FACT

In this issue of Molecular Cell, Engeholm etal.1 present cryo-EM structures of the chromatin remodeler Chd1 bound to a hexasome-nucleosome complex, an intermediate state during transcription either with or without FACT to restore the missing H2A-H2B dimer. These two binding modes reveal how Chd1 and FACT cooperate in nucleosome re-establishment during transcription.

The chromatin remodeler Chd1 supports MRX and Exo1 functions in resection of DNA double-strand breaks.

Repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) requires that the 5'-terminated DNA strands are resected to generate single-stranded DNA overhangs. This process is initiated by a short-range resection catalyzed by the MRX (Mre11-Rad50-Xrs2) complex, which is followed by a long-range step involving the nucleases Exo1 and Dna2. Here we show that the Saccharomyces cerevisiae ATP-dependent chromatin-remodeling protein Chd1 participates in both short- and long-range resection by promoting MRX and Exo1 association with the DSB ends. Furthermore, Chd1 reduces histone occupancy near the DSB ends and promotes DSB repair by HR. All these functions require Chd1 ATPase activity, supporting a role for Chd1 in the opening of chromatin at the DSB site to facilitate MRX and Exo1 processing activities.

Chd1 protects genome integrity at promoters to sustain hypertranscription in embryonic stem cells

Stem and progenitor cells undergo a global elevation of nascent transcription, or hypertranscription, during key developmental transitions involving rapid cell proliferation. The chromatin remodeler Chd1 mediates hypertranscription in pluripotent cells but its mechanism of action remains poorly understood. Here we report a novel role for Chd1 in protecting genome integrity at promoter regions by preventing DNA double-stranded break (DSB) accumulation in ES cells. Chd1 interacts with several DNA repair factors including Atm, Parp1, Kap1 and Topoisomerase 2β and its absence leads to an accumulation of DSBs at Chd1-bound Pol II-transcribed genes and rDNA. Genes prone to DNA breaks in Chd1 KO ES cells are longer genes with GC-rich promoters, a more labile nucleosomal structure and roles in chromatin regulation, transcription and signaling. These results reveal a vulnerability of hypertranscribing stem cells to accumulation of endogenous DNA breaks, with important implications for developmental and cancer biology.

FACT is recruited to the +1 nucleosome of transcribed genes and spreads in a Chd1-dependent manner

The histone chaperone FACT occupies transcribed regions where it plays prominent roles in maintaining chromatin integrity and preserving epigenetic information. How it is targeted to transcribed regions, however, remains unclear. Proposed models include docking on the RNA polymerase II (RNAPII) C-terminal domain (CTD), recruitment by elongation factors, recognition of modified histone tails, and binding partially disassembled nucleosomes. Here, we systematically test these and other scenarios in Saccharomyces cerevisiae and find that FACT binds transcribed chromatin, not RNAPII. Through a combination of high-resolution genome-wide mapping, single-molecule tracking, and mathematical modeling, we propose that FACT recognizes the+1 nucleosome, as it is partially unwrapped by the engaging RNAPII, and spreads to downstream nucleosomes aided by the chromatin remodeler Chd1. Our work clarifies how FACT interacts with genes, suggests a processive mechanism for FACT function, and provides a framework to further dissect the molecular mechanisms of transcription-coupled histone chaperoning.

Structural basis of nucleosome transcription mediated by Chd1 and FACT

Efficient transcription of RNA polymerase II (Pol II) through nucleosomes requires the help of various factors. Here we show biochemically that Pol II transcription through a nucleosome is facilitated by the chromatin remodeler Chd1 and the histone chaperone FACT when the elongation factors Spt4/5 and TFIIS are present. We report cryo-EM structures of transcribing Saccharomyces cerevisiae Pol II−Spt4/5−nucleosome complexes with bound Chd1 or FACT. In the first structure, Pol II transcription exposes the proximal histone H2A−H2B dimer that is bound by Spt5. Pol II has also released the inhibitory DNA-binding region of Chd1 that is poised to pump DNA toward Pol II. In the second structure, Pol II has generated a partially unraveled nucleosome that binds FACT, which excludes Chd1 and Spt5. These results suggest that Pol II progression through a nucleosome activates Chd1, enables FACT binding and eventually triggers transfer of FACT together with histones to upstream DNA.

Chromatin Remodeler CHD1 Forms Ternary Complexes with ADP and Pi Analogue which Mimic Transient States in ATPase Cycle

Chromatin remodeler Chd1 is one of the major types of ATP driven motor protein which moves along DNA, gaining energy from ATP hydrolysis and uses DNA as a ‘Rail’ to promote gene transcription by moving or ejecting the nucleosome. However, the motor mechanism by which this is done is still obscure at molecular level. In our study, we analyzed the properties of the Chd1-ADP-Pi analogues ternary complexes which mimic transient states in ATPase cycle for both the full length CHD1 (CHD1-FL) and catalytical motor domain of CHD1 (CHD1-MD) in order to clarify how ATP chemical energy transduced to motor activity.

Measurement of Nucleotide Hydrolysis Using Fluorescent Biosensors for Phosphate.

Assays for the detection of inorganic phosphate (Pi) are widely used to measure the activity of nucleotide hydrolyzing enzymes, such as ATPases and GTPases. The fluorescent biosensors for Pi, described here, are based on fluorescently labeled versions of E. coli phosphate-binding protein (PBP), which translates Pi binding into a large change in fluorescence intensity. In comparison with other Pi-detection systems, these biosensors are characterized by a high sensitivity (sub-micromolar Pi concentrations) and high time resolution (tens of milliseconds), and they are therefore particularly well suited for measurements of phosphate ester hydrolysis in real time. In this chapter, it is described how the Pi biosensors can be used to measure kinetics of ATPase and GTPase reactions, both under steady state and pre-steady state conditions. An example protocol is given for determining steady state kinetic parameters, Km and kcat, of the ATP-dependent chromatin remodeler Chd1, in a plate reader format. In addition, the measurement of Pi release kinetics under pre-steady state conditions is described, including a detailed experimental procedure for a singleturnover measurement of ATP hydrolysis by the ABC-type ATPase SufBC using rapid mixing.

The chromatin remodeler Chd1 regulates cohesin in budding yeast and humans

Chd1 is a chromatin remodeler that is involved in nucleosome positioning and transcription. Deletion of CHD1 is a frequent event in prostate cancer. The Structural Maintenance of Chromosome (SMC) complex cohesin mediates long-range chromatin interactions and is involved in maintaining genome stability. We provide new evidence that Chd1 is a regulator of cohesin. In the yeast S. cerevisiae, Chd1 is not essential for viability. We show that deletion of the gene leads to a defect in sister chromatid cohesion and in chromosome morphology. Chl1 is a non-essential DNA helicase that has been shown to regulate cohesin loading. Surprisingly, co-deletion of CHD1 and CHL1 results in an additive cohesion defect but partial suppression of the chromosome structure phenotype. We found that the cohesin regulator Pds5 is overexpressed when Chd1 and Chl1 are deleted. However, Pds5 expression is reduced to wild type levels when both genes are deleted. Finally, we show a correlation in the expression of CHD1 and cohesin genes in prostate cancer patients. Furthermore, we show that overexpression of cohesin subunits is correlated with the aggressiveness of the tumor. The biological roles of the interplay between Chd1, Chl1 and SMCs are discussed.

CHD1 Loss Alters AR Binding at Lineage-Specific Enhancers and Modulates Distinct Transcriptional Programs to Drive Prostate Tumorigenesis

Deletion of the gene encoding the chromatin remodeler CHD1 is among the most common alterations in prostate cancer (PCa); however, the tumor-suppressive functions of CHD1 and reasons for its tissue-specific loss remain undefined. We demonstrated that CHD1 occupied prostate-specific enhancers enriched for the androgen receptor (AR) and lineage-specific cofactors. Upon CHD1 loss, the AR cistrome was redistributed in patterns consistent with the oncogenic AR cistrome in PCa samples and drove tumor formation in the murine prostate. Notably, this cistrome shift was associated with a unique AR transcriptional signature enriched for pro-oncogenic pathways unique to this tumor subclass. Collectively, these data credential CHD1 as a tumor suppressor in the prostate that constrains AR binding/function to limit tumor progression.

TDP-43 Promotes Neurodegeneration by Impairing Chromatin Remodeling

Regulation of chromatin structure is critical for brain development and function. However, the involvement of chromatin dynamics in neurodegeneration is less well understood. Here we find, launching from Drosophila models of amyotrophic lateral sclerosis and frontotemporal dementia, that TDP-43 impairs the induction of multiple key stress genes required to protect from disease by reducing the recruitment of the chromatin remodeler Chd1 to chromatin. Chd1 depletion robustly enhances TDP-43-mediated neurodegeneration and promotes the formation of stress granules. Conversely, upregulation of Chd1 restores nucleosomal dynamics, promotes normal induction of protective stress genes, and rescues stress sensitivity of TDP-43-expressing animals. TDP-43-mediated impairments are conserved in mammalian cells, and, importantly, the human ortholog CHD2 physically interacts with TDP-43 and is strikingly reduced in level in temporal cortex of human patient tissue. These findings indicate that TDP-43-mediated neurodegeneration causes impaired chromatin dynamics that prevents appropriate expression of protective genes through compromised function of the chromatin remodeler Chd1/CHD2. Enhancing chromatin dynamics may be a treatment approach to amyotrophic lateral scleorosis (ALS)/frontotemporal dementia (FTD).

The ATP-dependent chromatin remodeler Chd1 is recruited by transcription elongation factors and maintains H3K4me3/H3K36me3 domains at actively transcribed and spliced genes.

The ATP-dependent chromatin remodeler Chd1 is recruited by transcription elongation factors and maintains H3K4me3/H3K36me3 domains at actively transcribed and spliced genes.

The ATP-dependent chromatin remodeler Chd1 is recruited by transcription elongation factors and maintains H3K4me3/H3K36me3 domains at actively transcribed and spliced genes.

Chd1 (Chromodomain Helicase DNA Binding Protein 1) is a conserved ATP-dependent chromatin remodeler that maintains the nucleosomal structure of chromatin, but the determinants of its specificity and its impact on gene expression are not well defined. To identify the determinants of Chd1 binding specificity in the yeast genome, we investigated Chd1 occupancy in mutants of several candidate factors. We found that several components of the PAF1 transcription elongation complex contribute to Chd1 recruitment to highly transcribed genes and identified Spt4 as a factor that appears to negatively modulate Chd1 binding to chromatin. We discovered that CHD1 loss alters H3K4me3 and H3K36me3 patterns throughout the yeast genome. Interestingly, the aberrant histone H3 methylation patterns were predominantly observed within 1 kb from the transcription start site, where both histone H3 methylation marks co-occur. A reciprocal change between the two marks was obvious in the absence of Chd1, suggesting a role for CHD1 in establishing or maintaining the boundaries of these largely mutually exclusive histone marks. Strikingly, intron-containing genes were most susceptible to CHD1 loss and exhibited a high degree of histone H3 methylation changes. Intron retention was significantly lower in the absence of CHD1, suggesting that CHD1 function as a chromatin remodeler could indirectly affect RNA splicing.

Impact of Dietary Interventions on Noncoding RNA Networks and mRNAs Encoding Chromatin-Related Factors

Dietary interventions dramatically affect metabolic disease and lifespan in various aging models. Here, we profiled liver microRNA (miRNA), coding, and long non-coding RNA (lncRNA) expression by high-throughput deep sequencing in mice across multiple energy intake and expenditure interventions. Strikingly, three dietary intervention network design patterns were uncovered: (1) lifespan-extending interventions largely repressed the expression of miRNAs, lncRNAs, and transposable elements; (2) protein-coding mRNAs with expression positively correlated with long lifespan are highly targeted by miRNAs; and (3) miRNA-targeting interactions mainly target chromatin-related functions. We experimentally validated miR-34a, miR-107, and miR-212-3p targeting of the chromatin remodeler Chd1 and further demonstrate that Chd1 knockdown mimics high-fat diet and aging-induced gene expression changes and activation of transposons. Our findingsdemonstrate lifespan-extending diets repress miRNA-chromatin remodeler interactions and safeguard against deregulated transcription induced by aging and lifespan shortening diets, events linked by microRNA, chromatin, and ncRNA crosstalk.

Impact of the Chromatin Remodeling Factor CHD1 on Gut Microbiome Composition of Drosophila melanogaster.

The composition of the intestinal microbiota of Drosophila has been studied in some detail in recent years. Environmental, developmental and host-specific genetic factors influence microbiome composition in the fly. Our previous work has indicated that intestinal bacterial load can be affected by chromatin-targeted regulatory mechanisms. Here we studied a potential role of the conserved chromatin assembly and remodeling factor CHD1 in the shaping of the gut microbiome in Drosophila melanogaster. Using high-throughput sequencing of 16S rRNA gene amplicons, we found that Chd1 deletion mutant flies exhibit significantly reduced microbial diversity compared to rescued control strains. Specifically, although Acetobacteraceae dominated the microbiota of both Chd1 wild-type and mutant guts, Chd1 mutants were virtually monoassociated with this bacterial family, whereas in control flies other bacterial taxa constituted ~20% of the microbiome. We further show age-linked differences in microbial load and microbiota composition between Chd1 mutant and control flies. Finally, diet supplementation experiments with Lactobacillus plantarum revealed that, in contrast to wild-type flies, Chd1 mutant flies were unable to maintain higher L. plantarum titres over time. Collectively, these data provide evidence that loss of the chromatin remodeler CHD1 has a major impact on the gut microbiome of Drosophila melanogaster.

Abstract A1-04: The role of chromatin remodeler CHD1 in genomic alterations and prostate cancer progression

Abstract The chromodomain helicase DNA (CHD) binding proteins have been implicated to play a role in genome stability. CHD1 is second only to PTEN as the most frequent homozygously deleted gene in the tumor genome of prostate cancer (PCa). To assess the role of CHD1 in genomic integrity and PCa progression, we performed a comprehensive survey of DNA copy number alterations (CNAs) in the tumor genome among a total of 611 patients from 5 independent cohorts and an association analysis of CHD1 deletion with cancer progression from low (Gleason 6) to high (Gleason ≥ 8) grade tumors. Our data revealed that the frequency of CHD1 deletion significantly increased as cancer progressing from low to intermediate and to high grades, suggesting a role of this CNA in cancer progression. Loss of CHD1 was significantly associated with homozygous deletions at other genomic locations in the tumor genome, and with deletion of MAP3K7. We next evaluated the in vitro and in vivo joint effects of knockdown of CHD1 and the collaborative gene MAP3K7 on tumorigenesis, cell proliferation (Ki67), tumor size, histopathological characteristics, and cancer progression, using shRNA and tissue recombination with rat urogenital mesenchyme and mouse prostate epithelial stem/progenitor cells grafted under the renal capsule and subcutaneous xenografts in nude mice. While suppressing CHD1 expression, in general, resulted in deceased growth of tumor cells vs. shControl cells, concurrently suppressing expression of both CHD1 and MAP3K7 promoted aggressive tumor growth and reduced survival. We finally explored the effect of knockdown of CHD1/MAP3K7 expression in stable cell lines derived human normal prostate epithelial cells and cancer cells on genomic rearrangement via CNA analysis. Although a wide range of CNAs was observed in different cell lines, some of them recapitulated the CNAs that were commonly observed in the tumors of human PCa. Collectively, these data suggest that loss of CHD1 leads to further evolution of cancer cells via genomic instability and combined loss of CHD1 and MAP3K7 drives the progression of PCa. Note: This abstract was not presented at the conference. Citation Format: Wennuan Liu, Lindsey Ulkus Rodrigues, Tao Li, Zheng Zhang, Leah Rider, Lina Romero, Lilly S. Zheng, William B. Isaacs, Scott Cramer, Jianfeng Xu. The role of chromatin remodeler CHD1 in genomic alterations and prostate cancer progression. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-04.

A genetic screen and transcript profiling reveal a shared regulatory program for Drosophila linker histone H1 and chromatin remodeler CHD1.

Chromatin structure and activity can be modified through ATP-dependent repositioning of nucleosomes and posttranslational modifications of core histone tails within nucleosome core particles and by deposition of linker histones into the oligonucleosome fiber. The linker histone H1 is essential in metazoans. It has a profound effect on organization of chromatin into higher-order structures and on recruitment of histone-modifying enzymes to chromatin. Here, we describe a genetic screen for modifiers of the lethal phenotype caused by depletion of H1 in Drosophila melanogaster. We identify 41 mis-expression alleles that enhance and 20 that suppress the effect of His1 depletion in vivo. Most of them are important for chromosome organization, transcriptional regulation, and cell signaling. Specifically, the reduced viability of H1-depleted animals is strongly suppressed by ubiquitous mis-expression of the ATP-dependent chromatin remodeling enzyme CHD1. Comparison of transcript profiles in H1-depleted and Chd1 null mutant larvae revealed that H1 and CHD1 have common transcriptional regulatory programs in vivo. H1 and CHD1 share roles in repression of numerous developmentally regulated and extracellular stimulus-responsive transcripts, including immunity-related and stress response-related genes. Thus, linker histone H1 participates in various regulatory programs in chromatin to alter gene expression.

Chd1 is essential for the high transcriptional output and rapid growth of the mouse epiblast.

The pluripotent mammalian epiblast undergoes unusually fast cell proliferation. This rapid growth is expected to generate a high transcriptional demand, but the underlying mechanisms remain unknown. We show here that the chromatin remodeler Chd1 is required for transcriptional output and development of the mouse epiblast. Chd1(-/-) embryos exhibit proliferation defects and increased apoptosis, are smaller than controls by E5.5 and fail to grow, to become patterned or to gastrulate. Removal of p53 allows progression of Chd1(-/-) mutants only to E7.0-8.0, highlighting the crucial requirement for Chd1 during early post-implantation development. Chd1(-/-) embryonic stem cells (ESCs) have a self-renewal defect and a genome-wide reduction in transcriptional output at both known mRNAs and intergenic transcripts. These transcriptional defects were only uncovered when cell number-normalized approaches were used, and correlate with a lower engagement of RNAP II with transcribed genes in Chd1(-/-) ESCs. We further show that Chd1 directly binds to ribosomal DNA, and that both Chd1(-/-) epiblast cells in vivo and ESCs in vitro express significantly lower levels of ribosomal RNA. In agreement with these findings, mutant cells in vivo and in vitro exhibit smaller and more elongated nucleoli. Thus, the RNA output by both Pol I and II is reduced in Chd1(-/-) cells. Our data indicate that Chd1 promotes a globally elevated transcriptional output required to sustain the distinctly rapid growth of the mouse epiblast.

The nucleosomal barrier to promoter escape by RNA polymerase II is overcome by the chromatin remodeler Chd1.

RNA polymerase II (PolII) transcribes RNA within a chromatin context, with nucleosomes acting as barriers to transcription. Despite these barriers, transcription through chromatin in vivo is highly efficient, suggesting the existence of factors that overcome this obstacle. To increase the resolution obtained by standard chromatin immunoprecipitation, we developed a novel strategy using micrococcal nuclease digestion of cross-linked chromatin. We find that the chromatin remodeler Chd1 is recruited to promoter proximal nucleosomes of genes undergoing active transcription, where Chd1 is responsible for the vast majority of PolII-directed nucleosome turnover. The expression of a dominant negative form of Chd1 results in increased stalling of PolII past the entry site of the promoter proximal nucleosomes. We find that Chd1 evicts nucleosomes downstream of the promoter in order to overcome the nucleosomal barrier and enable PolII promoter escape, thus providing mechanistic insight into the role of Chd1 in transcription and pluripotency. DOI: http://dx.doi.org/10.7554/eLife.02042.001.

CHD1 Contributes to Intestinal Resistance against Infection by P. aeruginosa in Drosophila melanogaster

Drosophila SNF2-type ATPase CHD1 catalyzes the assembly and remodeling of nucleosomal arrays in vitro and is involved in H3.3 incorporation in viin vivo during early embryo development. Evidence for a role as transcriptional regulator comes from its colocalization with elongating RNA polymerase II as well as from studies of fly Hsp70 transcription. Here we used microarray analysis to identify target genes of CHD1. We found a fraction of genes that were misregulated in Chd1 mutants to be functionally linked to Drosophila immune and stress response. Infection experiments using different microbial species revealed defects in host defense in Chd1-deficient adults upon oral infection with P. aeruginosa but not upon septic injury, suggesting a so far unrecognized role for CHD1 in intestinal immunity. Further molecular analysis showed that gut-specific transcription of antimicrobial peptide genes was overactivated in the absence of infection in Chd1 mutant flies. Moreover, microbial colonization of the intestine was elevated in Chd1 mutants and oral infection resulted in strong enrichment of bacteria in the body cavity indicating increased microbial passage across intestinal epithelia. However, we did not detect enhanced epithelial damage or alterations of the intestinal stem cell population. Collectively, our data provide evidence that intestinal resistance against infection by P. aeruginosa in Drosophila is linked to maintaining proper balance of gut-microbe interactions and that the chromatin remodeler CHD1 is involved in regulating this aspect.

The double life of Set1‐MLL in the initiation of transcription

The Set1/MLL1‐4 complexes represent the mammalian trithorax family of histone‐lysine methyltransferases. The Set1a/b‐containing complexes are responsible for H3K4 trimethylation, a modification which correlates with both actively transcribed and poised promoters. We show here, using an immobilized template assay, that Set1‐MLL is recruited to a pre‐initiation complex (PIC) on a chromatinized template by the model activator GAL4‐VP16. We observe that this recruitment can occur via a direct interaction with GAL4‐VP16, but is stimulated by the dual presence of the PIC components PAF and Mediator. To further understand the downstream consequences of Set1 function, we assembled synthetically modified H3K4me3 chromatin. The modification stimulated activator‐dependent in vitro transcription. Using a discovery‐based assay involving MuDPIT to identify H3K4‐specific factors captured on the immobilized template, we found that the chromatin remodeler CHD1 is enriched on H3K4me3 templates, but is recruited in an activator‐dependent manner, downstream of Mediator and MLL. In summary, preliminary findings suggest that the initial recruitment of Set1‐MLL is can occur both independent of or cooperatively with a functional PIC, the consequence of which is CHD1 recruitment and the stimulation of transcription. The work described above is supported by a grant from the NIH.