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Cellular differentiation requires extensive alterations in chromatin structure and function, which is elicited by the coordinated action of chromatin and transcription factors. By contrast with transcription factors, the roles of chromatin factors in differentiation have not been systematically characterized. Here, we combine bulk ex vivo and single-cell in vivo CRISPR screens to characterize the role of chromatin factor families in hematopoiesis. We uncover marked lineage specificities for 142 chromatin factors, revealing functional diversity among related chromatin factors (i.e. barrier-to-autointegration factor subcomplexes) as well as shared roles for unrelated repressive complexes that restrain excessive myeloid differentiation. Using epigenetic profiling, we identify functional interactions between lineage-determining transcription factors and several chromatin factors that explain their lineage dependencies. Studying chromatin factor functions in leukemia, we show that leukemia cells engage homeostatic chromatin factor functions to block differentiation, generating specific chromatin factor–transcription factor interactions that might be therapeutically targeted. Together, our work elucidates the lineage-determining properties of chromatin factors across normal and malignant hematopoiesis. Bulk ex vivo and single-cell in vivo CRISPR knockout screens are used to characterize 680 chromatin factors during mouse hematopoiesis, highlighting lineage-specific and normal and leukemia-specific functions.

Ras association (RalGDS/AF-6) domain family member 2 ( RASSF2 ) is a gene involved in the progression of several human cancers, including breast, colorectal and lung cancer. The aims of this study were to determine the hypermethylation of the gene in squamous cervical cancer and precursor lesions, along with that of RASSF1 and the recently described EPB41L3 , and to analyze the potential prognostic role of these genes. Methylation-specific PCR and bisulfite sequencing were used to analyze the methylation status of RASSF2 and EPB41L3 gene in 60 squamous cervical cancer, 76 cervical intraepithelial neoplasias grade III, 16 grade II, 14 grade I and 13 cases of normal tissue adjacent to cervical intraepithelial neoplasia. RASSF2 expression was evaluated by immunohistochemistry and the re-expression of RASSF2 and EPB41L3 was analyzed by quantitative reverse-transcription PCR in HeLa, SiHa, C33A and A431 cell lines treated with 5-aza-2′-deoxycytidine and/or trichostatin. RASSF1 hypermethylation and human papillomavirus type were also analyzed in all the cases by methylation-specific PCR and reverse line blot, respectively. RASSF2 hypermethylation was predominant in squamous cervical cancer (60.9%) compared with cervical intraepithelial neoplasias (4.2%) and was associated with a lower level of RASSF2 expression and vascular invasion in squamous cervical cancer. EPB41L3 and RASSF1 hypermethylations were also more frequent in cancer than in precursor lesions. Patients with RASSF2 hypermethylation had shorter survival time, independent of tumor stage (hazard ratio: 6.0; 95% confidence interval: 1.5–24.5). Finally, the expressions of RASSF2 and EPB41L3 were restored in several cell lines treated with 5-aza-2′-deoxycytidine. Taken together, our results suggest that RASSF2 potentially functions as a new tumor-suppressor gene that is inactivated through hypermethylation in cervical cancer and is related to the bad prognosis of these patients.

DNA methylation is essential for mammalian development and transcriptional repression of genes and retrotransposons during embryo development and in somatic cells. The patterns of DNA methylation are established by de novo DNA methyltransferases, which are regulated by developmental signalling and require access to chromatin. Besides DNA methyltransferases, other proteins have recently been implicated in DNA methylation, such as the ATP-dependent chromatin remodeler LSH. The absence of LSH in mouse embryos leads to defects in DNA methylation and development. In relation to this, mutations in LSH have been found to cause Immunodeficiency-Centromeric instability-Facial anomalies (ICF) syndrome. This syndrome is characterized by centromeric instability and CpG hypomethylation of centromeric satellite repeats, and is most often caused by mutations in the catalytic domain of the DNA methyltransferase DNMT3B. LSH is essential for developmentally programmed de novo DNA methylation of large chromosomal domains including promoters of protein coding genes and repetitive sequences. Importantly, fibroblasts derived from chromatin remodeling ATPase LSH-null mouse embryos, which lack DNA methylation at transposons and specific gene promoters, are capable of re-establishing normal patterns of DNA methylation and transcriptional silencing of misregulated genes upon re-expression of LSH. The ATP hydrolysis by LSH is essential for its function in gene silencing and de novo DNA methylation. However, the molecular mechanisms of LSH-dependent gene silencing and de novo DNA methylation are yet unclear. Here we use an inducible system that enables controlled expression of LSH in Lsh-null mouse embryonic fibroblasts (MEFs) to follow chromatin dynamics, transcriptional silencing and establishment of de novo DNA methylation. This conditionally reversible Lsh knockout cellular system allowed us to study the order of events occurring immediately after LSH restoration in MEF cell lines in order to elucidate the molecular mechanism of LSH-dependent gene silencing. We have demonstrated that LSH upon its restoration localises to the promoters of LSH-dependent loci leading to a mild decrease in the occupancy of H3, which reinforces the previously shown role of LSH as a chromatin remodeler. Simultaneously, there is removal of acetyl groups from H3 tails when LSH is bound to these target regions, which might be facilitated by the interaction of HDACs with LSH. The removal of H3Ac marks is followed by deposition of H3K9me2 by G9a/GLP histone methylases at the same time point when misregulated genes are silenced. This suggests that LSH creates a suitable substrate for G9a/GLP promoting gene silencing. Surprisingly, transcriptional repression occurs without acquisition of DNA methylation at the promoters of these loci. This order of events implies that LSH plays a role as a chromatin remodeler leading to changes in chromatin structure and modifications that facilitate epigenetic gene silencing without DNA methylation in the initial period when LSH is restored in MEF cell lines. Furthermore, deposition of H3K9me2 by the G9a/GLP complex is critical for silencing of specific genes, but not for repetitive elements such as IAPs. The histone modification H3K27me3 seems to play a transitory role in the silencing of IAP retrotransposons in the absence of G9a/GLP activity. In conclusion, this work has demonstrated that changes in chromatin modifications leading to a transcriptionally repressive chromatin state can be established in somatic cells by the chromatin remodeler LSH without acquisition of DNA methylation. This suggests that the primary role of LSH is to promote changes in chromatin structure and modifications that lead to gene silencing and not DNA methylation, which most likely occurs as a consequence of transcriptional silencing.