Analysis of Local Chromatin States Reveals Gene Transcription Potential during Mouse Neural Progenitor Cell Differentiation

HIRA complex presets transcriptional potential through coordinating depositions of the histone variants H3.3 and H2A.Z on the poised genes in mESCs

“…These results suggest that H3.3 and H2A.Z may function together with their chaperones to epigenetically regulate chromatin dynamics during transcriptional activation. To further investigate the crosstalk between HIRA-H3.3 pathway and H2A.Z deposition in chromatin structure, we performed time course digestion of reduced MNase (TC-rMNase) assay to analyze local chromatin accessibility ( Supplementary Figure S3A and S3B ) ( 51 ). As described in our previous study, TC-rMNase was an effective means to measure the local chromatin compaction using time-course digestion with mild enzyme activity of recombinant GST-MNase ( 51 ).…”

“…To further investigate the crosstalk between HIRA-H3.3 pathway and H2A.Z deposition in chromatin structure, we performed time course digestion of reduced MNase (TC-rMNase) assay to analyze local chromatin accessibility ( Supplementary Figure S3A and S3B ) ( 51 ). As described in our previous study, TC-rMNase was an effective means to measure the local chromatin compaction using time-course digestion with mild enzyme activity of recombinant GST-MNase ( 51 ). Here we simplified that method to collect mononucleosomes only digested with 2 and 4 minutes, of which a theoretically and preferentially high ratio derives from active and bivalent regions.…”

“…The detail steps of this procedure were performed as described previously ( 51 ). In briefly, cells were counted and collected under the native conditions.…”

“…In contrast, knockdown of H2A.Z or SRCAP did not obviously affect the H3.3 incorporation, supporting the upstream function of HIRA complex in this regulatory process. In addition, through partial chromatin digestion using our GST (glutathione S -transferase) tagged micrococcal nuclease followed by deep sequencing (GST-MNase-seq) and ATAC-seq assays ( 50 , 51 ), we showed that H2A.Z incorporation mediated by HIRA complex coupling with SRACP pathway contributed to the maintenance of high-order chromatin compaction at gene promoters. Furthermore, functional enrichment analysis found that the genes that could not be activated under tRA induction in HIRA complex deleted cell lines were significantly enriched in transcriptional regulation during neuronal development, which was largely supported by the phenotypic and molecular verifications.…”

Transcriptome Analysis and Intraspecific Variation in Spanish Fir (Abies pinsapo Boiss.)

“…These proteins have been widely reported to mediate transcription through posttranslational histone modifications [ 72 ] and cytosine methylation of specific DNA sequence [ 73 ] processes. Therefore, different chromatin states and genome DNA methylation patterns could affect gene transcription, as has been described in multiple eukaryotic organisms [ 74 , 75 , 76 , 77 , 78 ], since these modifications play important roles in the regulation of chromatin condensation and DNA accessibility [ 79 ]. All these phenomena could also explain the differences observed in the differential expression of several transcription factors ( Figure 2 ) that could be involved in the differential gene responses observed.…”