Depletion of the Chromatin Remodeler CHD4 Sensitizes AML Blasts to Genotoxic Agents and Reduces Tumor Formation

Chromodomain Helicase DNA-Binding Protein 4 (CHD4) is an ATPase that alters the phasing of nucleosomes on DNA and has recently been implicated in DNA double stranded break (DSB) repair. Here, we show that depletion of CHD4 in Acute Myeloid Leukemia (AML) blasts induces a global relaxation of chromatin that renders cells more susceptible to DSB formation, while concurrently impeding their repair. Furthermore, CHD4 depletion renders AML blasts more sensitive both in vitro and in vivo to genotoxic agents used in clinical therapy: daunorubicin (DNR) and cytarabine (ara-C). Sensitization to DNR and ara-C is mediated in part by activation of the ATM pathway, which is preliminarily activated by a Tip60-dependent mechanism in response to chromatin relaxation and further activated by genotoxic-agent induced DSBs. This sensitization preferentially affects AML cells, as CHD4 depletion in normal CD34+ hematopoetic progenitors does not increase their susceptibility to DNR or ara-C. Unexpectedly, we found that CHD4 is necessary for maintaining the tumor formatting behavior of AML cells, as CHD4 depletion severely restricted the ability of AML cells to form xenografts in mice and colonies in soft agar. Taken together, these results provide evidence for CHD4 as a novel therapeutic target whose inhibition has the potential to enhance the effectiveness of genotoxic agents used in AML therapy

STRUCTURE AND FUNCTION STUDIES OF METHYL-CPG BINDING DOMAIN PROTEINS AND THEIR COMPLEXES

DNA methylation is an epigenetic mechanism of transcriptional silencing of increasing interest for treating human disease. Methyl-CpG binding domain (MBD) proteins recognize 5 methylcytosines (mC) primarily in symmetrical CpG (mCG) dinucleotides. Seven proteins comprise the MBD family, MBD1-6 and MeCP2. MeCP2 is primarily expressed in the brain and plays a critical role in neuron maturation, as mutations disrupting its function account for up to 80% of Rett Syndrome cases. MBD2/3 associates with the nucleosome remodeling and deacetylase complex (NuRD) and modulates gene expression through alteration of the chromatin architecture surrounding the mC mark. In these studies, we examine the behavior of the MBDs of MeCP2 and MBD2, in addition to further characterizing the protein-protein interactions between subunits of NuRD. Recent work suggests the primary effects of MeCP2 on gene expression in the developing mammalian brain are mediated by binding asymmetrically methylated and hydroxymethylated CpA (h/mCA) dinucleotides. This work establishes that the MeCP2 MBD binds mCA with high affinity in a strand specific and orientation dependent manner. This preference is specific to MeCP2, as the MBD2 MBD does not show high affinity or methyl-specific binding to mCA. Introduction of the Rett Syndrome-associated mutations T158M, R106W and P101S destabilized the MeCP2 MBD and lessened recognition of mCG and mCA equally. Finally, hydroxymethylation of a high affinity mCA site did not dramatically change binding properties, however hemi-hydroxylation of the same cytosine in mCG significantly decreased affinity. We suggest a model for MeCP2 recognition of mCA and for hydroxymethylation as an epigenetic switch to redistribute MeCP2 among mCG and mCA loci. Blocking recruitment of NuRD by MBD2 restores expression of developmentally silenced fetal hemoglobin and aberrantly silenced tumor suppressor genes. Additionally, knockdown of the NuRD helicase, CHD4, results in cancer cells growth arrest and increased sensitivity to DNA damage. Therefore, targeting MBD2-NuRD presents a promising avenue for treating β-hemoglobinopathies and cancer. Towards understanding the recruitment of the NuRD components to the complex, this study characterizes the GATA-like zinc-finger domains of the NuRD components GATAD2A and MTA2. We propose a model of NuRD in which MTA2 binds DNA and GATAD2A serves to bridge MBD2 and CHD4.Doctor of Philosoph

Depletion of the chromatin remodeler CHD4 sensitizes AML blasts to genotoxic agents and reduces tumor formation

Chromodomain helicase DNA-binding protein 4 (CHD4) is an ATPase that alters the phasing of nucleosomes on DNA and has recently been implicated in DNA double-stranded break (DSB) repair. Here, we show that depletion of CHD4 in acute myeloid leukemia (AML) blasts induces a global relaxation of chromatin that renders cells more susceptible to DSB formation, while concurrently impeding their repair. Furthermore, CHD4 depletion renders AML blasts more sensitive both in vitro and in vivo to genotoxic agents used in clinical therapy: daunorubicin (DNR) and cytarabine (ara-C). Sensitization to DNR and ara-C is mediated in part by activation of the ataxia-telangiectasia mutated pathway, which is preliminarily activated by a Tip60-dependent mechanism in response to chromatin relaxation and further activated by genotoxic agent–induced DSBs. This sensitization preferentially affects AML cells, as CHD4 depletion in normal CD34+ hematopoietic progenitors does not increase their susceptibility to DNR or ara-C. Unexpectedly, we found that CHD4 is necessary for maintaining the tumor-forming behavior of AML cells, as CHD4 depletion severely restricted the ability of AML cells to form xenografts in mice and colonies in soft agar. Taken together, these results provide evidence for CHD4 as a novel therapeutic target whose inhibition has the potential to enhance the effectiveness of genotoxic agents used in AML therapy

Methylation specific targeting of a chromatin remodeling complex from sponges to humans

DNA cytosine methylation and methyl-cytosine binding domain (MBD) containing proteins are found throughout all vertebrate species studied to date. However, both the presence of DNA methylation and pattern of methylation varies among invertebrate species. Invertebrates generally have only a single MBD protein, MBD2/3, that does not always contain appropriate residues for selectively binding methylated DNA. Therefore, we sought to determine whether sponges, one of the most ancient extant metazoan lineages, possess an MBD2/3 capable of recognizing methylated DNA and recruiting the associated nucleosome remodeling and deacetylase (NuRD) complex. We find that Ephydatia muelleri has genes for each of the NuRD core components including an EmMBD2/3 that selectively binds methylated DNA. NMR analyses reveal a remarkably conserved binding mode, showing almost identical chemical shift changes between binding to methylated and unmethylated CpG dinucleotides. In addition, we find that EmMBD2/3 and EmGATAD2A/B proteins form a coiled-coil interaction known to be critical for the formation of NuRD. Finally, we show that knockdown of EmMBD2/3 expression disrupts normal cellular architecture and development of E. muelleri. These data support a model in which the MBD2/3 methylation-dependent functional role emerged with the earliest multicellular organisms and has been maintained to varying degrees across animal evolution

Crystal structure analysis reveals Pseudomonas PilY1 as an essential calcium-dependent regulator of bacterial surface motility

Several bacterial pathogens require the “twitching” motility produced by filamentous type IV pili (T4P) to establish and maintain human infections. Two cytoplasmic ATPases function as an oscillatory motor that powers twitching motility via cycles of pilus extension and retraction. The regulation of this motor, however, has remained a mystery. We present the 2.1 Å resolution crystal structure of the Pseudomonas aeruginosa pilus-biogenesis factor PilY1, and identify a single site on this protein required for bacterial translocation. The structure reveals a modified β-propeller fold and a distinct EF-hand-like calcium-binding site conserved in pathogens with retractile T4P. We show that preventing calcium binding by PilY1 using either an exogenous calcium chelator or mutation of a single residue disrupts Pseudomonas twitching motility by eliminating surface pili. In contrast, placing a lysine in this site to mimic the charge of a bound calcium interferes with motility in the opposite manner—by producing an abundance of nonfunctional surface pili. Our data indicate that calcium binding and release by the unique loop identified in the PilY1 crystal structure controls the opposing forces of pilus extension and retraction. Thus, PilY1 is an essential, calcium-dependent regulator of bacterial twitching motility

Quantitative modelling predicts the impact of DNA methylation on RNA polymerase II traffic

Patterns of gene expression are primarily determined by proteins that locally enhance or repress transcription. While many transcription factors target a restricted number of genes, others appear to modulate transcription levels globally. An example is MeCP2, an abundant methylated-DNA binding protein that is mutated in the neurological disorder Rett Syndrome. Despite much research, the molecular mechanism by which MeCP2 regulates gene expression is not fully resolved. Here we integrate quantitative, multidimensionalexperimental analysis and mathematical modelling to indicate that MeCP2 is a novel type of global transcriptional regulator whose binding to DNA creates "slow sites" in gene bodies. We hypothesise that waves of slowed-down RNA polymerase II formed behind these sites travel backward and indirectly affect initiation, reminiscent of defect-induced shock waves in non-equilibrium physics transport models. This mechanism differs from conventional gene regulation mechanisms, which often involve direct modulation of transcription initiation. Our findings point to a genome-wide function of DNA methylation that may account for the reversibility of Rett syndrome in mice. Moreover, our combined theoretical and experimental approach provides a general method for understanding how global gene expression patterns are choreographed

A lexicon of DNA modifications: their roles in embryo development and the germline

5-methylcytosine (5mC) on CpG dinucleotides has been viewed as the major epigenetic modification in eukaryotes for a long time. Apart from 5mC, additional DNA modifications have been discovered in eukaryotic genomes. Many of these modifications are thought to be solely associated with DNA damage. However, growing evidence indicates that some base modifications, namely 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), 5-carboxylcytosine (5caC), and N6-methadenine (6mA), may be of biological relevance, particularly during early stages of embryo development. Although abundance of these DNA modifications in eukaryotic genomes can be low, there are suggestions that they cooperate with other epigenetic markers to affect DNA-protein interactions, gene expression, defense of genome stability and epigenetic inheritance. Little is still known about their distribution in different tissues and their functions during key stages of the animal lifecycle. This review discusses current knowledge and future perspectives of these novel DNA modifications in the mammalian genome with a focus on their dynamic distribution during early embryonic development and their potential function in epigenetic inheritance through the germ line

Refinement of the subunit interaction network within the nucleosome remodelling and deacetylase (NuRD) complex

The nucleosome remodelling and deacetylase (NuRD) complex is essential for the development of complex animals. NuRD has roles in regulating gene expression and repairing damaged DNA. The complex comprises at least six proteins with two or more paralogues of each protein routinely identified when the complex is purified from cell extracts. To understand the structure and function of NuRD, a map of direct subunit interactions is needed. Dozens of published studies have attempted to define direct inter-subunit connectivities. We propose that conclusions reported in many such studies are in fact ambiguous for one of several reasons. First, the expression of many NuRD subunits in bacteria is unlikely to lead to folded, active protein. Second, interaction studies carried out in cells that contain endogenous NuRD complex can lead to false positives through bridging of target proteins by endogenous components. Combining existing information on NuRD structure with a protocol designed to minimize false positives, we report a conservative and robust interaction map for the NuRD complex. We also suggest a 3D model of the complex that brings together the existing data on the complex. The issues and strategies discussed herein are also applicable to the analysis of a wide range of multi-subunit complexes.Micrococcal nuclease (MNase), EC 3.1.31.1; histone deacetylase (HDAC), EC 3.5.1.98