To identify chromatin mechanisms of neuronal differentiation, we characterized chromatin accessibility and gene expression in cerebellar granule neurons (CGNs) of the developing mouse. We used DNase-seq to map accessibility of cis-regulatory elements and RNA-seq to profile transcript abundance across postnatal stages of neuronal differentiation in vivo and in culture. We observed thousands of chromatin accessibility changes as CGNs differentiated and verified by H3K27ac ChIP-seq, reporter gene assays, and CRISPR-mediated activation that many of these regions function as neuronal enhancers. Motif discovery within differentially accessible chromatin regions suggested a novel role for the Zic family of transcription factors in CGN maturation. We confirmed the association of Zic with these elements by ChIP-seq, and demonstrated by knockdown that Zic1/2 are required to coordinate mature neuronal gene expression patterns. Together these data reveal chromatin dynamics at thousands of gene regulatory elements that facilitate gene expression patterns necessary for neuronal differentiation and function.

BRCA1 is a well-known DNA repair pathway component and a tissue-specific tumor suppressor. However, its role in hematopoiesis is uncertain. Here we report that a cohort of patients heterozygous for BRCA1 mutations experienced more hematopoietic toxicity from chemotherapy than those with BRCA2 mutations. To test whether this reflects a requirement for BRCA1 in hematopoiesis, we generated mice with Brca1 mutations in hematopoietic cells. Mice homozygous for a null Brca1 mutation in the embryonic hematopoietic system (Vav1-iCre;Brca1F22–24/F22-24) developed hematopoietic defects in early adulthood that included reduced hematopoietic stem cells (HSCs). Although mice homozygous for a huBRCA1 knock-in allele (Brca1BRCA1/BRCA1) were normal, mice with a mutant huBRCA1/5382insC allele and a null allele (Mx1-Cre;Brca1F22-24/5382insC), had severe hematopoietic defects marked by a complete loss of hematopoietic stem and progenitor cells. Our data show that Brca1 is necessary for HSC maintenance and normal hematopoiesis, and that distinct mutations lead to different degrees of hematopoietic dysfunction.

The histone H3 lysine 27 (H3K27) demethylase Kdm6b (Jmjd3) can promote cellular differentiation, however its physiological functions in neurons remain to be fully determined. We studied the expression and function of Kdm6b in differentiating granule neurons of the developing postnatal mouse cerebellum. At postnatal day 7, Kdm6b is expressed throughout the layers of the developing cerebellar cortex, but its expression is upregulated in newborn cerebellar granule neurons (CGNs). Atoh1-Cre mediated conditional knockout of Kdm6b in CGN precursors either alone or in combination with Kdm6a did not disturb the gross morphological development of the cerebellum. Furthermore, RNAi-mediated knockdown of Kdm6b in cultured CGN precursors did not alter the induced expression of early neuronal marker genes upon cell cycle exit. By contrast, knockdown of Kdm6b significantly impaired the induction of a mature neuronal gene expression program, which includes gene products required for functional synapse maturation. Loss of Kdm6b also impaired the ability of Brain-Derived Neurotrophic Factor (BDNF) to induce expression of Grin2c and Tiam1 in maturing CGNs. Taken together, these data reveal a previously unknown role for Kdm6b in the postmitotic stages of CGN maturation and suggest that Kdm6b may work, at least in part, by a transcriptional mechanism that promotes gene sensitivity to regulation by BDNF.

PURPOSE Hereditary cancer genetic testing can inform personalized medical management for individuals at increased cancer risk. However, many variants in cancer predisposition genes are individually rare, and traditional tools may be insufficient to evaluate pathogenicity. This analysis presents data on variant classification and reclassification over a 20-year period. PATIENTS AND METHODS This is a retrospective analysis of > 1.9 million individuals who received hereditary cancer genetic testing from a single clinical laboratory (March 1997 to December 2017). Variant classification included review of evidence from traditional tools (eg, population frequency databases, literature) and laboratory-developed tools (eg, novel statistical methods, in-house RNA analysis) by a multidisciplinary expert committee. Variants may have been reclassified more than once and with more than one line of evidence. RESULTS In this time period, 62,842 unique variants were observed across 25 cancer predisposition genes, and 2,976 variants were reclassified. Overall, 82.1% of reclassification events were downgrades (eg, variant of uncertain significance [VUS] to benign), and 17.9% were upgrades (eg, VUS to pathogenic). Among reclassified variants, 82.8% were initially classified as VUS, and 47.5% were identified in ≤ 20 individuals (allele frequency ≤ 0.001%). Laboratory-developed tools were used in 72.3% of variant reclassification events, which affected > 600,000 individuals. More than 1.3 million patients were identified as carrying a variant that was reclassified within this 20-year time period. CONCLUSION The variant classification program used by the laboratory evaluated here enabled the reclassification of variants that were individually rare. Laboratory-developed tools were a key component of this program and were used in the majority of reclassifications. This demonstrates the importance of using robust and novel tools to reclassify rare variants to appropriately inform personalized medical management.

Enzymes that regulate histone lysine methylation play important roles in neuronal differentiation, but little is known about their contributions to activity-regulated gene transcription in differentiated neurons. We characterized activity-regulated expression of lysine demethylases and lysine methyltransferases in the hippocampus of adult male mice following pilocarpine-induced seizure. Pilocarpine drove a 20-fold increase in mRNA encoding the histone H3 lysine27-specific demethylase Kdm6b selectively in granule neurons of the dentate gyrus, and this induction was recapitulated in cultured hippocampal neurons by bicuculline and 4-aminopyridine (Bic+4AP) stimulation of synaptic activity. Because activity-regulated gene expression is highly correlated with neuronal survival, we tested the requirement for Kdm6b expression in Bic+4AP induced preconditioning of neuronal survival. Prior exposure to Bic+4AP promoted neuronal survival in control neurons upon growth factor withdrawal, however this effect was ablated when we knocked down Kdm6b expression. Loss of Kdm6b did not disrupt activity-induced expression of most genes, including that of a gene set previously established to promote neuronal survival in this assay. However using bioinformatic analysis of RNA sequencing data, we discovered that Kdm6b knockdown neurons showed impaired inducibility of a discrete set of genes annotated for their function in inflammation. These data reveal a novel function for Kdm6b in activity-regulated neuronal survival, and they suggest that activity- and Kdm6b-dependent regulation of inflammatory gene pathways may serve as an adaptive pro-survival response to increased neuronal activity.

Deficiency of huntingtin-interacting protein 1 (Hip1) results in degenerative phenotypes. Here we generated a deficiency allele where a floxed transcriptional stop cassette and a human cDNA were knocked into intron 1 of the mouse locus.-mediated germ line excision of the stop cassette resulted in expression of HIP1 and rescue of the knockout phenotype.-mediated excision led to HIP1 expression in spleen, kidney and liver, and also rescued the phenotype. In contrast, -mediated, brain-specific HIP1 expression did not rescue the phenotype. Metabolomics and microarrays of several knockout tissues identified low phosphocholine (PC) levels and low glycerophosphodiester phosphodiesterase domain containing 3 (Gdpd3) gene expression. Since Gdpd3 has lysophospholipase D activity that results in the formation of choline, a precursor of PC, downregulation could lead to the low PC levels. To test whether contributes to the deficiency phenotype, we generated knockout mice. Double knockout of and worsened the Hip1 phenotype. This suggests that Gdpd3 compensates for Hip1 loss. More-detailed knowledge of how deficiency leads to low PC will improve our understanding of HIP1 in choline metabolism in normal and disease states.

SummaryBoth BRCA1 and CREBBP are tumor suppressor genes that are important for hematopoiesis. We have previously shown that mouse Brca1 is essential for hematopoietic stem cell (HSC) viability. In contrast to Brca1 deficiency, which results in pancytopenia, we report here that Crebbp deficiency results in myeloproliferation associated with an increase of splenic HSCs as well as a lethal systemic inflammatory disorder (LD50 = 86 days). To investigate the interaction of these two proteins in hematopoiesis, we generated double Crebbp/Brca1 knockout mice (DKOs). To our surprise, DKOs had accelerated bone marrow failure compared with Brca1-deficient mice and this was associated with an even shorter lifespan (LD50 = 88.5 versus 33 days). Furthermore, Crebbp or Brca1 heterozygosity influenced the hematopoietic phenotype associated with complete deficiency of Brca1 or Crebbp, respectively. We also observed lower BRCA1 protein levels in hematopoietic tissues when CREBBP is absent. Collectively, these data suggest Crebbp and Brca1 functionally interact to maintain normal hematopoiesis.

47Deficiency of huntingtin interacting protein 1 (Hip1) results in degenerative phenotypes. 48Here we generated a Hip1 deficiency allele where a floxed transcriptional stop-cassette 49 and a human HIP1 cDNA were knocked-in to intron 1 of mouse Hip1 locus. CMV-Cre-50 mediated germline excision of the stop-cassette resulted in expression of HIP1 and rescue 51 of the Hip1 knockout phenotype. Mx1-Cre-mediated excision led to HIP1 expression in 52 spleen, kidney and liver, and also rescued the phenotype. In contrast, GFAP-Cre-53 mediated HIP1 expression in brain did not rescue the phenotype. Metabolomics and 54 microarrays of several Hip1 knockout tissues identified low phosphocholine (PC) levels 55 and low Glycerophosphodiester Phosphodiesterase Domain Containing 3 (Gdpd3) 56 expression. Since Gdpd3 has lysophospholipase D activity that results in the formation of 57 choline, a precursor of PC, Gdpd3 downregulation could lead to the low PC levels. To 58 test if Gdpd3 contributes to the Hip1 deficiency phenotype, we generated Gdpd3 59 knockout mice. Double knockout of Gdpd3 and Hip1 worsened the Hip1 phenotype. This 60 suggests that Gdpd3 compensates for Hip1 loss. More detailed knowledge of how Hip1 61 deficiency leads to low PC will improve our understanding of HIP1 in choline 62 metabolism in normal and disease states. 63 64 65 amplified from pcDNA3-hHip1 plasmid. The IRES (~0.6 kb) and eGFP (~0.9 kb) 128 sequences were amplified from pIRES2-AcGFP1 and pEGFP-N1 plasmid, respectively. 129The final vector was obtained by standard molecular cloning. Aside from the 130 homology arms, the final vector also contains a Lox-Stop-Lox cassette (~1.5 kb), mHip1 131 partial intron1+ partial exon 2, hHip1 cDNA, IRES, EGFP + polyA, Frt sequences flanking 132 the Neo expression cassette (the neo cassette was used for positive selection of the 133 electroporated ES cells), and a DTA expression cassette (for negative selection of the ES 134