Overarching control of autophagy and DNA damage response by CHD6 revealed by modeling a rare human pathology.

Members of the chromodomain-helicase-DNA binding (CHD) protein family are chromatin remodelers implicated in human pathologies, with CHD6 being one of its least studied members. We discovered a de novo CHD6 missense mutation in a patient clinically presenting the rare Hallermann-Streiff syndrome (HSS). We used genome editing to generate isogenic iPSC lines and model HSS in relevant cell types. By combining genomics with functional in vivo and in vitro assays, we show that CHD6 binds a cohort of autophagy and stress response genes across cell types. The HSS mutation affects CHD6 protein folding and impairs its ability to recruit co-remodelers in response to DNA damage or autophagy stimulation. This leads to accumulation of DNA damage burden and senescence-like phenotypes. We therefore uncovered a molecular mechanism explaining HSS onset via chromatin control of autophagic flux and genotoxic stress surveillance.

CHD7 regulates cardiovascular development through ATP-dependent and -independent activities.

CHD7 encodes an ATP-dependent chromatin remodeling factor. Mutation of this gene causes multiple developmental disorders, including CHARGE (Coloboma of the eye, Heart defects, Atresia of the choanae, Retardation of growth/development, Genital abnormalities, and Ear anomalies) syndrome, in which conotruncal anomalies are the most prevalent form of heart defects. How CHD7 regulates conotruncal development remains unclear. In this study, we establish that deletion of Chd7 in neural crest cells (NCCs) causes severe conotruncal defects and perinatal lethality, thus providing mouse genetic evidence demonstrating that CHD7 cell-autonomously regulates cardiac NCC development, thereby clarifying a long-standing controversy in the literature. Using transcriptomic analyses, we show that CHD7 fine-tunes the expression of a gene network that is critical for cardiac NCC development. To gain further molecular insights into gene regulation by CHD7, we performed a protein-protein interaction screen by incubating recombinant CHD7 on a protein array. We find that CHD7 directly interacts with several developmental disorder-mutated proteins including WDR5, a core component of H3K4 methyltransferase complexes. This direct interaction suggested that CHD7 may recruit histone-modifying enzymes to target loci independently of its remodeling functions. We therefore generated a mouse model that harbors an ATPase-deficient allele and demonstrates that mutant CHD7 retains the ability to recruit H3K4 methyltransferase activity to its targets. Thus, our data uncover that CHD7 regulates cardiovascular development through ATP-dependent and -independent activities, shedding light on the etiology of CHD7-related congenital disorders. Importantly, our data also imply that patients carrying a premature stop codon versus missense mutations will likely display different molecular alterations; these patients might therefore require personalized therapeutic interventions.

The DNA repair protein SHPRH is a nucleosome-stimulated ATPase and a nucleosome-E3 ubiquitin ligase.

BACKGROUND: Maintenance of genome integrity during DNA replication is crucial to the perpetuation of all organisms. In eukaryotes, the bypass of DNA lesions by the replication machinery prevents prolonged stalling of the replication fork, which could otherwise lead to greater damages such as gross chromosomal rearrangements. Bypassing DNA lesions and subsequent repair are accomplished by the activation of DNA damage tolerance pathways such as the template switching (TS) pathway. In yeast, the RAD5 (Radiation-sensitive 5) protein plays a crucial role in initiating the TS pathway by catalyzing the polyubiquitination of PCNA (Proliferation Cell Nuclear Antigen). Likewise, one of the mammalian RAD5-homologs, SHPRH (SNF2, histone linker, PHD, RING, helicase) mediates PCNA polyubiquitination. To date, the study of SHPRH enzymatic functions has been limited to this modification. It is therefore unclear how SHPRH carries out its function in DNA repair. Moreover, how this protein regulates gene transcription at the enzymatic level is also unknown. RESULTS: Given that SHPRH harbors domains found in chromatin remodeling proteins, we investigated its biochemical properties in the presence of nucleosomal substrates. We find that SHPRH binds equally well to double-stranded (ds) DNA and to nucleosome core particles, however, like ISWI and CHD-family remodelers, SHPRH shows a strong preference for nucleosomes presenting extranucleosomal DNA. Moreover, nucleosomes but not dsDNA strongly stimulate the ATPase activity of SHPRH. Intriguingly, unlike typically observed with SNF2-family enzymes, ATPase activity does not translate into conventional nucleosome remodeling, under standard assay conditions. To test whether SHPRH can act as a ubiquitin E3 ligase for nucleosomes, we performed a screen using 26 E2-conjugating enzymes. We uncover that SHPRH is a potent nucleosome E3-ubiquitin-ligase that can function with at least 7 different E2s. Mass spectrometry analyses of products generated in the presence of the UBE2D1-conjugating enzyme reveal that SHPRH can catalyze the formation of polyubiquitin linkages that are either branched or associated with the recruitment of DNA repair factors, as well as linkages involved in proteasomal degradation. CONCLUSIONS: We propose that, in addition to polyubiquitinating PCNA, SHPRH promotes DNA repair or transcriptional regulation in part through chromatin ubiquitination. Our study sets a biochemical framework for studying other RAD5- and RAD16-related protein functions through the ubiquitination of nucleosomes.