Regulation of HOXA2 gene expression by the ATP‐dependent chromatin remodeling enzyme CHD8

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/116349/1/feb2s0014579310000414.pd

A Human BRCA2 Complex Containing a Structural DNA Binding Component Influences Cell Cycle Progression

AbstractGermline mutations of the human BRCA2 gene confer susceptibility to breast cancer. Although the function of the BRCA2 protein remains to be determined, murine cells homozygous for BRCA2 inactivation display chromosomal aberrations. We have isolated a 2 MDa BRCA2-containing complex and identified a structural DNA binding component, designated as BR CA2-A ssociated F actor 35 (BRAF35). BRAF35 contains a nonspecific DNA binding HMG domain and a kinesin-like coiled coil domain. Similar to BRCA2, BRAF35 mRNA expression levels in mouse embryos are highest in proliferating tissues with high mitotic index. Strikingly, nuclear staining revealed a close association of BRAF35/BRCA2 complex with condensed chromatin coincident with histone H3 phosphorylation. Importantly, antibody microinjection experiments suggest a role for BRCA2/BRAF35 complex in modulation of cell cycle progression

A core–BRAF35 complex containing histone deacetylase mediates repression of neuronal-specific genes

BRAF35, a structural DNA-binding protein, initially was identified as a component of a large BRCA2-containing complex. Biochemical analysis revealed the presence of a smaller core–BRAF35 complex devoid of BRCA2. Here we report the isolation of a six-subunit core–BRAF35 complex with the capacity to deacetylate histones, termed the BRAF–histone deacetylase complex (BHC), from human cells. BHC contains polypeptides reminiscent of the chromatin-remodeling complexes SWI/SNF and NuRD (nucleosome remodeling and deacetylating). Similar to NuRD, BHC contains an Mi2-like subunit, BHC80, and a PHD zinc-finger subunit as well as histone deacetylases 1/2 and an MTA-like subunit, the transcriptional corepressor CoREST. We show that BHC mediates repression of neuron-specific genes through the cis-regulatory element known as the repressor element 1 or neural restrictive silencer (RE1/NRS). Chromatin-immunoprecipitation experiments demonstrate the recruitment of BHC by the neuronal repressor REST. Expression of BRAF35 containing a single point mutation in the HMG domain of the protein abrogated REST-mediated transcriptional repression. These results demonstrate a role for core–BRAF35-containing complex in the regulation of neuron-specific genes through modulation of the chromatin structure

Inhibition of Transcription by the Trimeric Cyclin-dependent Kinase 7 Complex

Cyclin-dependent kinase 7 (CDK7) can be isolated as a subunit of a trimeric kinase complex functional in activation of the mitotic promoting factor. In this study, we demonstrate that the trimeric cdk-activating kinase (CAK) acts as a transcriptional repressor of class II promoters and show that repression results from CAK impeding the entry of RNA polymerase II and basal transcription factor IIF into a competent preinitiation complex. This repression is independent of CDK7 kinase activity. We find that the p36/MAT1 subunit of CAK is required for transcriptional repression and the repression is independent of the promoter used. Our results demonstrate a central role for CAK in regulation of messenger RNA synthesis by either inhibition of RNA polymerase II-catalyzed transcription or stimulation of transcription through association with basal transcription repair factor IIH

Functional Interplay between Histone Demethylase and Deacetylase Enzymes

Histone deacetylase (HDAC) inhibitors are a promising class of anticancer agents for the treatment of solid and hematological malignancies. The precise mechanism by which HDAC inhibitors mediate their effects on tumor cell growth, differentiation, and/or apoptosis is the subject of intense research. Previously we described a family of multiprotein complexes that contain histone deacetylase 1/2 (HDAC1/2) and the histone demethylase BHC110 (LSD1). Here we show that HDAC inhibitors diminish histone H3 lysine 4 (H3K4) demethylation by BHC110 in vitro. In vivo analysis revealed an increased H3K4 methylation concomitant with inhibition of nucleosomal deacetylation by HDAC inhibitors. Reconstitution of recombinant complexes revealed a functional connection between HDAC1 and BHC110 only when nucleosomal substrates were used. Importantly, while the enzymatic activity of BHC110 is required to achieve optimal deacetylation in vitro, in vivo analysis following ectopic expression of an enzymatically dead mutant of BHC110 (K661A) confirmed the functional cross talk between the demethylase and deacetylase enzymes. Our studies not only reveal an intimate link between the histone demethylase and deacetylase enzymes but also identify histone demethylation as a secondary target of HDAC inhibitors

A family of chromatin remodeling factors related to Williams syndrome transcription factor

Chromatin remodeling complexes have been implicated in the disruption or reformation of nucleosomal arrays resulting in modulation of transcription, DNA replication, and DNA repair. Here we report the isolation of WCRF, a new chromatin-remodeling complex from HeLa cells. WCRF is composed of two subunits, WCRF135, the human homolog of Drosophila ISWI, and WCRF180, a protein related to the Williams syndrome transcription factor. WCRF180 is a member of a family of proteins sharing a putative heterochromatin localization domain, a PHD finger, and a bromodomain, prevalent in factors involved in regulation of chromatin structure

Reply to Correspondence on “Synergy and Antagonism between Allosteric and Active-Site Inhibitors of Abl Tyrosine Kinase”

Manley and co-workers provide data demonstrating that, at super-pharmacological concentrations (300 μM), a ternary complex between Abl, asciminib, and ATP-competitive inhibitors is possible. The work in our manuscript concerns the interplay of asciminib (and GNF-2) with ATP-competitive inhibitors at pharmacologically relevant concentrations (Cmax=1.6–3.7 μM for asciminib). Manley and co-workers do not question any of the studies that we reported, nor do they provide explanations for how our work fits into their preferred model. Herein, we consider the data presented by Manley and co-workers. In addition, we provide new data supporting the findings in our Communication. Asciminib and ATP-competitive inhibitors do not simultaneously bind Abl at pharmacologically relevant concentrations unless the conformation selectivity for both ligands is matched.At pharmacologically relevant concentrations, asciminib and clinical adenosine triphosphate (ATP) kinase inhibitors cannot simultaneously bind to Abl kinase. Manley and co-workers correspond that at saturating concentrations (i.e., concentrations that are not achievable in a human), asciminib and dasatinib can simultaneously bind to Abl kinase.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/175174/1/ange202209518-sup-0001-misc_information.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/175174/2/ange202209518.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/175174/3/ange202209518_am.pd

Synergy and Antagonism between Allosteric and Active‐Site Inhibitors of Abl Tyrosine Kinase

Allosteric inhibitors of Abl kinase are being explored in the clinic, often in combination with ATP‐site inhibitors of Abl kinase. However, there are conflicting data on whether both ATP‐competitive inhibitors and myristoyl‐site allosteric inhibitors can simultaneously bind Abl kinase. Here, we determine whether there is synergy or antagonism between ATP‐competitive inhibitors and allosteric inhibitors of Abl. We observe that clinical ATP‐competitive inhibitors are not synergistic with allosteric ABL inhibitors, however, conformation‐selective ATP‐site inhibitors that modulate the global conformation of Abl can afford synergy. We demonstrate that kinase conformation is the key driver to simultaneously bind two compounds to Abl kinase. Finally, we explore the interaction of allosteric and conformation selective ATP‐competitive inhibitors in a series of biochemical and cellular assays.We describe a better understanding of the interaction between allosteric and ATP‐competitive inhibitors of Abl. Using a protease accessibility assay we developed, we determined the global conformation of Abl when bound to Abl inhibitors. We found that synergy between ATP‐competitive and allosteric inhibitors of Abl required a „matched“ kinase conformation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/169317/1/ange202105351_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/169317/2/ange202105351.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/169317/3/ange202105351-sup-0001-misc_information.pd