Mapping histone variant genomic distribution: Exploiting SNAP-tag labeling to follow the dynamics of incorporation of H3 variants.

In the eukaryotic cell nucleus, in addition to the genomic information, chromatin organization provides an additional set of information which is more versatile and associates with distinct cell identities. In particular, the marking of the nucleosomes by a choice of specific histone variants can potentially confer distinct functional properties critical for genome function and stability. To understand how this unique marking operates we need to access to the genomic distribution of each variant. A general approach based on ChIP-Seq, relies on the specific isolation of DNA bound to the variant of interest, usually using cross-linked material and specific antibodies. The availability of reliable specific antibodies recognizing with high affinity crosslinked antigen represents a limitation. Here, we describe an experimental approach exploiting a tag fused to the protein of interest. The chose protein is a histone variant and we use native conditions for the selective capture of the histone variant in a nucleosome. Most importantly, we describe how to use a particular labeling system, with a SNAP tag enabling to specifically capture nucleosomes comprising newly synthesized histones. This method allows to follow the newly deposited histone variant at various times thereby offering a unique opportunity to evaluate the dynamics of histone deposition genome wide. We describe the method here for H3 variant, but it can be adapted to any histone variant with the appropriate fused tag to address genome wide a turn-over associated to the biological context of interest.

Regulation of replicative histone RNA metabolism by the histone chaperone ASF1.

In S phase, duplicating and assembling the whole genome into chromatin requires upregulation of replicative histone gene expression. Here, we explored how histone chaperones control histone production in human cells to ensure a proper link with chromatin assembly. Depletion of the ASF1 chaperone specifically decreases the pool of replicative histones both at the protein and RNA levels. The decrease in their overall expression, revealed by total RNA sequencing (RNA-seq), contrasted with the increase in nascent/newly synthesized RNAs observed by 4sU-labeled RNA-seq. Further inspection of replicative histone RNAs showed a 3' end processing defect with an increase of pre-mRNAs/unprocessed transcripts likely targeted to degradation. Collectively, these data argue for a production defect of replicative histone RNAs in ASF1-depleted cells. We discuss how this regulation of replicative histone RNA metabolism by ASF1 as a "chaperone checkpoint" fine-tunes the histone dosage to avoid unbalanced situations deleterious for cell survival.

HIRA-dependent boundaries between H3 variants shape early replication in mammals.

The lack of a consensus DNA sequence defining replication origins in mammals has led researchers to consider chromatin as a means to specify these regions. However, to date, there is no mechanistic understanding of how this could be achieved and maintained given that nucleosome disruption occurs with each fork passage and with transcription. Here, by genome-wide mapping of the de novo deposition of the histone variants H3.1 and H3.3 in human cells during S phase, we identified how their dual deposition mode ensures a stable marking with H3.3 flanked on both sides by H3.1. These H3.1/H3.3 boundaries correspond to the initiation zones of early origins. Loss of the H3.3 chaperone HIRA leads to the concomitant disruption of H3.1/H3.3 boundaries and initiation zones. We propose that the HIRA-dependent deposition of H3.3 preserves H3.1/H3.3 boundaries by protecting them from H3.1 invasion linked to fork progression, contributing to a chromatin-based definition of early replication zones.

CENP-A Subnuclear Localization Pattern as Marker Predicting Curability by Chemoradiation Therapy for Locally Advanced Head and Neck Cancer Patients.

Effective biomarkers predictive of the response to treatments are key for precision medicine. This study identifies the staining pattern of the centromeric histone 3 variant, CENP-A, as a predictive biomarker of locoregional disease curability by chemoradiation therapy. We compared by imaging the subnuclear distribution of CENP-A in normal and tumoral tissues, and in a retrospective study in biopsies of 62 locally advanced head and neck squamous cell carcinoma (HNSCC) patients treated by chemoradiation therapy. We looked for predictive factors of locoregional disease control and patient's survival, including CENP-A patterns, Ki67, HPV status and anisokaryosis. In different normal tissues, we reproducibly found a CENP-A subnuclear pattern characterized by CENP-A clusters both localized at the nuclear periphery and regularly spaced. In corresponding tumors, both features are lost. In locally advanced HNSCC, a specific CENP-A pattern identified in pretreatment biopsies predicts definitive locoregional disease control after chemoradiation treatment in 96% (24/25) of patients (OR = 17.6 CI 95% [2.6; 362.8], p = 0.002), independently of anisokaryosis, Ki67 labeling or HPV status. The characteristics of the subnuclear pattern of CENP-A in cell nuclei revealed by immunohistochemistry could provide an easy to use a reliable marker of disease curability by chemoradiation therapy in locally advanced HNSCC patients.

High-resolution visualization of H3 variants during replication reveals their controlled recycling.

DNA replication is a challenge for the faithful transmission of parental information to daughter cells, as both DNA and chromatin organization must be duplicated. Replication stress further complicates the safeguard of epigenome integrity. Here, we investigate the transmission of the histone variants H3.3 and H3.1 during replication. We follow their distribution relative to replication timing, first in the genome and, second, in 3D using super-resolution microscopy. We find that H3.3 and H3.1 mark early- and late-replicating chromatin, respectively. In the nucleus, H3.3 forms domains, which decrease in density throughout replication, while H3.1 domains increase in density. Hydroxyurea impairs local recycling of parental histones at replication sites. Similarly, depleting the histone chaperone ASF1 affects recycling, leading to an impaired histone variant landscape. We discuss how faithful transmission of histone variants involves ASF1 and can be impacted by replication stress, with ensuing consequences for cell fate and tumorigenesis.

Whole genome and transcriptome analysis of a novel AML cell line with a normal karyotype.

Acute myeloid leukemia (AML) occurs when hematopoietic progenitor cells acquire genetic defects blocking the regulation of normal growth and differentiation. Although recurrent translocations have been identified in AML, almost half of adult AML patients present with a normal karyotype (NK-AML). While cell line models exist to study AML, they frequently have abnormal/unstable karyotypes, while primary cells from NK-AML patients are difficult to maintain in vitro. Here we provide a thorough molecular characterization of a recently established cell line, CG-SH, which has normal cytogenetics, representing a useful new model for NK-AML. Using high-throughput DNA sequencing, we first defined the genetic background of this cell line. In addition to identifying potentially deleterious SNVs in genes relevant to AML, we also found insertions in both GATA2 and EZH2, two genes previously linked to AML. We further characterized the growth of this model system in vitro with a cytokine mix that promotes faster cell growth. We assessed gene expression changes after the addition of cytokines to the culture media and found differential expression in genes implicated in proliferation, apoptosis and differentiation. Our results provide a detailed molecular characterization of genetic defects in this cell line derived from an NK-AML patient.

A universal RNA polymerase II CTD cycle is orchestrated by complex interplays between kinase, phosphatase, and isomerase enzymes along genes.

Transcription by RNA polymerase II (RNAPII) is coupled to mRNA processing and chromatin modifications via the C-terminal domain (CTD) of its largest subunit, consisting of multiple repeats of the heptapeptide YSPTSPS. Pioneering studies showed that CTD serines are differentially phosphorylated along genes in a prescribed pattern during the transcription cycle. Genome-wide analyses challenged this idea, suggesting that this cycle is not uniform among different genes. Moreover, the respective role of enzymes responsible for CTD modifications remains controversial. Here, we systematically profiled the location of the RNAPII phosphoisoforms in wild-type cells and mutants for most CTD modifying enzymes. Together with results of in vitro assays, these data reveal a complex interplay between the modifying enzymes, and provide evidence that the CTD cycle is uniform across genes. We also identify Ssu72 as the Ser7 phosphatase and show that proline isomerization is a key regulator of CTD dephosphorylation at the end of genes.

DSIF and RNA polymerase II CTD phosphorylation coordinate the recruitment of Rpd3S to actively transcribed genes.

Histone deacetylase Rpd3 is part of two distinct complexes: the large (Rpd3L) and small (Rpd3S) complexes. While Rpd3L targets specific promoters for gene repression, Rpd3S is recruited to ORFs to deacetylate histones in the wake of RNA polymerase II, to prevent cryptic initiation within genes. Methylation of histone H3 at lysine 36 by the Set2 methyltransferase is thought to mediate the recruitment of Rpd3S. Here, we confirm by ChIP-Chip that Rpd3S binds active ORFs. Surprisingly, however, Rpd3S is not recruited to all active genes, and its recruitment is Set2-independent. However, Rpd3S complexes recruited in the absence of H3K36 methylation appear to be inactive. Finally, we present evidence implicating the yeast DSIF complex (Spt4/5) and RNA polymerase II phosphorylation by Kin28 and Ctk1 in the recruitment of Rpd3S to active genes. Taken together, our data support a model where Set2-dependent histone H3 methylation is required for the activation of Rpd3S following its recruitment to the RNA polymerase II C-terminal domain.

The euchromatic and heterochromatic landscapes are shaped by antagonizing effects of transcription on H2A.Z deposition.

A role for variant histone H2A.Z in gene expression is now well established but little is known about the mechanisms by which it operates. Using a combination of ChIP-chip, knockdown and expression profiling experiments, we show that upon gene induction, human H2A.Z associates with gene promoters and helps in recruiting the transcriptional machinery. Surprisingly, we also found that H2A.Z is randomly incorporated in the genome at low levels and that active transcription antagonizes this incorporation in transcribed regions. After cessation of transcription, random H2A.Z quickly reappears on genes, demonstrating that this incorporation utilizes an active mechanism. Within facultative heterochromatin, we observe a hyper accumulation of the variant histone, which might be due to the lack of transcription in these regions. These results show how chromatin structure and transcription can antagonize each other, therefore shaping chromatin and controlling gene expression.

Comparison of promoter activities for efficient expression into human B cells and haematopoietic progenitors with adenovirus Ad5/F35.

Adenoviral gene transfer into human B lymphocytes and haematopoietic progenitors would allow the characterization of their function on cellular growth, differentiation and survival. Efficient gene expression is however strongly dependent on the promoter used. In this study, we investigated the relative strength of various promoters by following and measuring the expression of the reporter gene EYFP in human peripheral B lymphocytes, cord blood CD34(+) cells and the megakaryocytic cell line M-07e. The murine PGK promoter provided the best level of transgene expression in CD34(+) cells among the four promoters tested, followed closely by the CMV promoter, and to a lesser extend by a CMV promoter with a beta-globin/IgG chimeric intron, whereas the human CD40 promoter provided the lowest levels of expression. In contrast, the strongest promoters in B lymphocytes were the two CMV promoters. Surprisingly, even the best promoters were unable to induce transgene expression in more than 75-80% of the primary B and CD34(+) cells, even though 100% of the cells were infected. Finally and in contrast to retroviruses, only a minority of B lymphocytes and CD34(+) cells were able to induce the transcription of IRES-containing bicistronic expression cassettes from adenovirus.