Interaction of CCR4-NOT with EBF1 regulates gene-specific transcription and mRNA stability in B lymphopoiesis.

Transcription factor EBF1 (early B-cell factor 1) regulates early B-cell differentiation by poising or activating lineage-specific genes and repressing genes associated with alternative cell fates. To identify proteins that regulate the diverse functions of EBF1, we used SILAC (stable isotope labeling by amino acids in cell culture)-based mass spectrometry of proteins associated with endogenous EBF1 in pro-B cells. This analysis identified most components of the multifunctional CCR4-NOT complex, which regulates transcription and mRNA degradation. CNOT3 interacts with EBF1, and we identified histidine 240 in EBF1 as a critical residue for this interaction. Complementation of Ebf1-/- progenitors with EBF1H240A revealed a partial block of pro-B-cell differentiation and altered expression of specific EBF1 target genes that show either reduced transcription or increased mRNA stability. Most deregulated EBF1 target genes show normal occupancy by EBF1H240A, but we also detected genes with altered occupancy, suggesting that the CCR4-NOT complex affects multiple activities of EBF1. Mice with conditional Cnot3 inactivation recapitulate the block of early B-cell differentiation, which we found to be associated with an impaired autoregulation of Ebf1 and reduced expression of pre-B-cell receptor components. Thus, the interaction of the CCR4-NOT complex with EBF1 diversifies the function of EBF1 in a context-dependent manner and may coordinate transcriptional and post-transcriptional gene regulation.

Pol5 is an essential ribosome biogenesis factor required for 60S ribosomal subunit maturation in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, more than 250 trans-acting factors are involved in the maturation of 40S and 60S ribosomal subunits. The expression of most of these factors is transcriptionally coregulated to ensure correct ribosome production under a wide variety of environmental and intracellular conditions. Here, we identified the essential nucleolar Pol5 protein as a novel trans-acting factor required for the synthesis of 60S ribosomal subunits. Pol5 weakly and/or transiently associates with early to medium pre-60S ribosomal particles. Depletion of and temperature-sensitive mutations in Pol5 result in a deficiency of 60S ribosomal subunits and accumulation of half-mer polysomes. Both processing of 27SB pre-rRNA to mature 25S rRNA and release of pre-60S ribosomal particles from the nucle(ol)us to the cytoplasm are impaired in the Pol5-depleted strain. Moreover, we identified the genes encoding ribosomal proteins uL23 and eL27A as multicopy suppressors of the slow growth of a temperature-sensitive pol5 mutant. These results suggest that Pol5 could function in ensuring the correct folding of 25S rRNA domain III; thus, favoring the correct assembly of these two ribosomal proteins at their respective binding sites into medium pre-60S ribosomal particles. Pol5 is homologous to the human tumor suppressor Myb-binding protein 1A (MYBBP1A). However, expression of MYBBP1A failed to complement the lethal phenotype of a pol5 null mutant strain though interfered with 60S ribosomal subunit biogenesis.

The prefoldin complex regulates chromatin dynamics during transcription elongation.

Transcriptional elongation requires the concerted action of several factors that allow RNA polymerase II to advance through chromatin in a highly processive manner. In order to identify novel elongation factors, we performed systematic yeast genetic screening based on the GLAM (Gene Length-dependent Accumulation of mRNA) assay, which is used to detect defects in the expression of long transcription units. Apart from well-known transcription elongation factors, we identified mutants in the prefoldin complex subunits, which were among those that caused the most dramatic phenotype. We found that prefoldin, so far involved in the cytoplasmic co-translational assembly of protein complexes, is also present in the nucleus and that a subset of its subunits are recruited to chromatin in a transcription-dependent manner. Prefoldin influences RNA polymerase II the elongation rate in vivo and plays an especially important role in the transcription elongation of long genes and those whose promoter regions contain a canonical TATA box. Finally, we found a specific functional link between prefoldin and histone dynamics after nucleosome remodeling, which is consistent with the extensive network of genetic interactions between this factor and the machinery regulating chromatin function. This study establishes the involvement of prefoldin in transcription elongation, and supports a role for this complex in cotranscriptional histone eviction.

Integration of stress signals by homeodomain interacting protein kinases.

The family of homeodomain interacting protein kinases (HIPKs) consists of four related kinases, HIPK1 to HIPK4. These serine/threonine kinases are evolutionary conserved and derive from the yeast kinase Yak1. The largest group of HIPK phosphorylation substrates is represented by transcription factors and chromatin-associated regulators of gene expression, thus transferring HIPK-derived signals into changes of gene expression programs. The HIPKs mainly function as regulators of developmental processes and as integrators of a wide variety of stress signals. A number of conditions representing precarious situations, such as DNA damage, hypoxia, reactive oxygen intermediates and metabolic stress affect the function of HIPKs. The kinases function as integrators for these stress signals and feed them into many different downstream effector pathways that serve to cope with these precarious situations. HIPKs do not function as essential core components in the different stress signaling pathways, but rather serve as modulators of signal output and as connectors of different stress signaling pathways. Their central role as signaling hubs with the ability to shape many downstream effector pathways frequently implies them in proliferative diseases such as cancer or fibrosis.

The distribution of active RNA polymerase II along the transcribed region is gene-specific and controlled by elongation factors.

In order to study the intragenic profiles of active transcription, we determined the relative levels of active RNA polymerase II present at the 3'- and 5'-ends of 261 yeast genes by run-on. The results obtained indicate that the 3'/5' run-on ratio varies among the genes studied by over 12 log(2) units. This ratio seems to be an intrinsic characteristic of each transcriptional unit and does not significantly correlate with gene length, G + C content or level of expression. The correlation between the 3'/5' RNA polymerase II ratios measured by run-on and those obtained by chromatin immunoprecipitation is poor, although the genes encoding ribosomal proteins present exceptionally low ratios in both cases. We detected a subset of elongation-related factors that are important for maintaining the wild-type profiles of active transcription, including DSIF, Mediator, factors related to the methylation of histone H3-lysine 4, the Bur CDK and the RNA polymerase II subunit Rpb9. We conducted a more detailed investigation of the alterations caused by rpb9Delta to find that Rpb9 contributes to the intragenic profiles of active transcription by influencing the probability of arrest of RNA polymerase II.

Yeast genetic analysis reveals the involvement of chromatin reassembly factors in repressing HIV-1 basal transcription.

Rebound of HIV viremia after interruption of anti-retroviral therapy is due to the small population of CD4+ T cells that remain latently infected. HIV-1 transcription is the main process controlling post-integration latency. Regulation of HIV-1 transcription takes place at both initiation and elongation levels. Pausing of RNA polymerase II at the 5' end of HIV-1 transcribed region (5'HIV-TR), which is immediately downstream of the transcription start site, plays an important role in the regulation of viral expression. The activation of HIV-1 transcription correlates with the rearrangement of a positioned nucleosome located at this region. These two facts suggest that the 5'HIV-TR contributes to inhibit basal transcription of those HIV-1 proviruses that remain latently inactive. However, little is known about the cell elements mediating the repressive role of the 5'HIV-TR. We performed a genetic analysis of this phenomenon in Saccharomyces cerevisiae after reconstructing a minimal HIV-1 transcriptional system in this yeast. Unexpectedly, we found that the critical role played by the 5'HIV-TR in maintaining low levels of basal transcription in yeast is mediated by FACT, Spt6, and Chd1, proteins so far associated with chromatin assembly and disassembly during ongoing transcription. We confirmed that this group of factors plays a role in HIV-1 postintegration latency in human cells by depleting the corresponding human orthologs with shRNAs, both in HIV latently infected cell populations and in particular single-integration clones, including a latent clone with a provirus integrated in a highly transcribed gene. Our results indicate that chromatin reassembly factors participate in the establishment of the equilibrium between activation and repression of HIV-1 when it integrates into the human genome, and they open the possibility of considering these factors as therapeutic targets of HIV-1 latency.

Regulon-specific control of transcription elongation across the yeast genome.

Transcription elongation by RNA polymerase II was often considered an invariant non-regulated process. However, genome-wide studies have shown that transcriptional pausing during elongation is a frequent phenomenon in tightly-regulated metazoan genes. Using a combination of ChIP-on-chip and genomic run-on approaches, we found that the proportion of transcriptionally active RNA polymerase II (active versus total) present throughout the yeast genome is characteristic of some functional gene classes, like those related to ribosomes and mitochondria. This proportion also responds to regulatory stimuli mediated by protein kinase A and, in relation to cytosolic ribosomal-protein genes, it is mediated by the silencing domain of Rap1. We found that this inactive form of RNA polymerase II, which accumulates along the full length of ribosomal protein genes, is phosphorylated in the Ser5 residue of the CTD, but is hypophosphorylated in Ser2. Using the same experimental approach, we show that the in vivo-depletion of FACT, a chromatin-related elongation factor, also produces a regulon-specific effect on the expression of the yeast genome. This work demonstrates that the regulation of transcription elongation is a widespread, gene class-dependent phenomenon that also affects housekeeping genes.

Genome-wide analysis of factors affecting transcription elongation and DNA repair: a new role for PAF and Ccr4-not in transcription-coupled repair.

RNA polymerases frequently deal with a number of obstacles during transcription elongation that need to be removed for transcription resumption. One important type of hindrance consists of DNA lesions, which are removed by transcription-coupled repair (TC-NER), a specific sub-pathway of nucleotide excision repair. To improve our knowledge of transcription elongation and its coupling to TC-NER, we used the yeast library of non-essential knock-out mutations to screen for genes conferring resistance to the transcription-elongation inhibitor mycophenolic acid and the DNA-damaging agent 4-nitroquinoline-N-oxide. Our data provide evidence that subunits of the SAGA and Ccr4-Not complexes, Mediator, Bre1, Bur2, and Fun12 affect transcription elongation to different extents. Given the dependency of TC-NER on RNA Polymerase II transcription and the fact that the few proteins known to be involved in TC-NER are related to transcription, we performed an in-depth TC-NER analysis of a selection of mutants. We found that mutants of the PAF and Ccr4-Not complexes are impaired in TC-NER. This study provides evidence that PAF and Ccr4-Not are required for efficient TC-NER in yeast, unraveling a novel function for these transcription complexes and opening new perspectives for the understanding of TC-NER and its functional interconnection with transcription elongation.

Broad Transcriptomic Impact of Sorafenib and Its Relation to the Antitumoral Properties in Liver Cancer Cells.

Hepatocellular carcinoma (HCC) is one of the most frequent and essentially incurable cancers in its advanced stages. The tyrosine kinase inhibitor Sorafenib (Sfb) remains the globally accepted treatment for advanced HCC. However, the extent of its therapeutic benefit is limited. Sfb exerts antitumor activity through its cytotoxic, anti-proliferative and pro-apoptotic roles in HCC cells. To better understand the molecular mechanisms underlying these effects, we used RNA sequencing to generate comprehensive transcriptome profiles of HepG2 and SNU423, hepatoblastoma- (HB) and HCC-derived cell lines, respectively, following a Sfb treatment at a pharmacological dose. This resulted in similar alterations of gene expression in both cell lines. Genes functionally related to membrane trafficking, stress-responsible and unfolded protein responses, circadian clock and activation of apoptosis were predominantly upregulated, while genes involved in cell growth and cycle, DNA replication and repair, ribosome biogenesis, translation initiation and proteostasis were downregulated. Our results suggest that Sfb causes primary effects on cellular stress that lead to upregulation of selective responses to compensate for its negative effect and restore homeostasis. No significant differences were found specifically affecting each cell line, indicating the robustness of the Sfb mechanism of action despite the heterogeneity of liver cancer. We discuss our results on terms of providing rationalization for possible strategies to improve Sfb clinical outcomes.

Effect of Vitamin D on Graft-versus-Host Disease.

The different cell subsets of the immune system express the vitamin D receptor (VDR). Through the VDR, vitamin D exerts different functions that influence immune responses, as previously shown in different preclinical models. Based on this background, retrospective studies explored the impacts of vitamin D levels on the outcomes of patients undergoing allogeneic hematopoietic stem-cell transplantation, showing that vitamin D deficiency is related to an increased risk of complications, especially graft-versus-host disease. These results were confirmed in a prospective cohort trial, although further studies are required to confirm this data. In addition, the role of vitamin D on the treatment of hematologic malignancies was also explored. Considering this dual effect on both the immune systems and tumor cells of patients with hematologic malignancies, vitamin D might be useful in this setting to decrease both graft-versus-host disease and relapse rates.

Combined treatment of graft versus host disease using donor regulatory T cells and ruxolitinib.

Donor derived regulatory T lymphocytes and the JAK1/2 kinase inhibitor ruxolitinib are currently being evaluated as therapeutic options in the treatment of chronic graft versus host disease (cGvHD). In this work, we aimed to determine if the combined use of both agents can exert a synergistic effect in the treatment of GvHD. For this purpose, we studied the effect of this combination both in vitro and in a GvHD mouse model. Our results show that ruxolitinib favors the ratio of thymic regulatory T cells to conventional T cells in culture, without affecting the suppressive capacity of these Treg. The combination of ruxolitinib with Treg showed a higher efficacy as compared to each single treatment alone in our GvHD mouse model in terms of GvHD incidence, severity and survival without hampering graft versus leukemia effect. This beneficial effect correlated with the detection in the bone marrow of recipient mice of the infused donor allogeneic Treg after the adoptive transfer.

Upregulation of CD38 expression on multiple myeloma cells by novel HDAC6 inhibitors is a class effect and augments the efficacy of daratumumab.

Multiple myeloma (MM) is incurable, so there is a significant unmet need for effective therapy for patients with relapsed or refractory disease. This situation has not changed despite the recent approval of the anti-CD38 antibody daratumumab, one of the most potent agents in MM treatment. The efficiency of daratumumab might be improved by combining it with synergistic anti-MM agents. We therefore investigated the potential of the histone deacetylase (HDAC) inhibitor ricolinostat to up-regulate CD38 on MM cells, thereby enhancing the performance of CD38-specific therapies. Using quantitative reverse transcription polymerase chain reaction and flow cytometry, we observed that ricolinostat significantly increases CD38 RNA levels and CD38 surface expression on MM cells. Super-resolution microscopy imaging of MM cells by direct stochastic optical reconstruction microscopy confirmed this rise with molecular resolution and revealed homogeneous distribution of CD38 molecules on the cell membrane. Particularly important is that combining ricolinostat with daratumumab induced enhanced lysis of MM cells. We also evaluated next-generation HDAC6 inhibitors (ACY-241, WT-161) and observed similar increase of CD38 levels suggesting that the upregulation of CD38 expression on MM cells by HDAC6 inhibitors is a class effect. This proof-of-concept illustrates the potential benefit of combining HDAC6 inhibitors and CD38-directed immunotherapy for MM treatment.

Delayed administration of ixazomib modifies the immune response and prevents chronic graft-versus-host disease.

In this study, we aimed to modify the immune response in the long term after allogeneic bone marrow transplantation (allo-BMT) by using the proteasome inhibitor ixazomib (IXZ) at the late stages of the post-transplant period. This approach facilitated the immune reconstitution after transplantation. IXZ significantly prolonged survival and decreased the risk of chronic graft-versus-host disease (cGvHD) in two different murine models without hampering the graft-versus-leukemia (GvL) effect, as confirmed by bioluminescence assays. Remarkably, the use of IXZ was related to an increase of regulatory T cells both in peripheral blood and in the GvHD target organs and a decrease of effector donor T cells. Regarding B cells, IXZ treated mice had faster recovery of B cells in PB and of pre-pro-B cells in the bone marrow. Mice receiving ixazomib had a lower number of neutrophils in the GvHD target organs as compared to the vehicle group. In summary, delayed administration of IXZ ameliorated cGvHD while preserving GvL and promoted a pro-tolerogenic immune response after allo-BMT.

Vitamin D Modifies the Incidence of Graft-versus-Host Disease after Allogeneic Stem Cell Transplantation Depending on the Vitamin D Receptor (VDR) Polymorphisms.

PURPOSE: The biologically active metabolite of vitamin D3, 1,25-dihydroxyvitamin D3 (vit D), has immunoregulatory properties via binding vitamin D receptor (VDR). In a prospective trial, we previously reported a reduction in the incidence of chronic GvHD (cGvHD) among patients who received vit D after allogeneic stem cell transplantation (allo-HSCT; Clinical Trials.gov: NCT02600988). Here we analyze the role of patients and donors' VDR SNPs on the immunomodulatory effect of vit D. PATIENTS AND METHODS: Patients undergoing allo-HSCT were included in a prospective phase I/II clinical trial (Alovita) in three consecutive cohorts: control (without vit D), low-dose (1,000 IU/day), and high-dose (5,000 IU/day) groups. Vit D was given from day -5 until +100 after transplant. Genotyping of four SNPs of the VDR gene, FokI, BsmI, ApaI, and TaqI, were performed using TaqMan SNP genotyping assays. RESULTS: We observed a decrease in the incidence of overall cGvHD at 1 year after allo-HSCT depending on the use or not of vit D among patients with FokI CT genotype (22.5% vs 80%, P = 0.0004) and among those patients without BsmI/ApaI/TaqI ATC haplotype (22.2% vs 68.8%, P = 0.0005). In a multivariate analysis, FokI CT genotype significantly influenced the risk of cGvHD in patients treated with vit D as compared with the control group (HR 0.143, P interaction < 0.001). CONCLUSIONS: Our results show that the immunomodulatory effect of vit D depends on the VDR SNPs, and patients carrying the FokI CT genotype display the highest benefit from receiving vit D after allo-HSCT.

Formaldehyde-assisted Isolation of Regulatory Elements to Measure Chromatin Accessibility in Mammalian Cells.

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly defined states of chromatin organization. The dynamic architecture of the nucleus is controlled by multiple mechanisms and shapes the transcriptional output programs. It is, therefore, important to determine locus-specific chromatin accessibility in a reliable fashion that is preferably independent from antibodies, which can be a potentially confounding source of experimental variability. Chromatin accessibility can be measured by various methods, including the Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) assay, that allow the determination of general chromatin accessibility in a relatively low number of cells. Here we describe a FAIRE protocol that allows simple, reliable, and fast identification of genomic regions with a low protein occupancy. In this method, the DNA is covalently bound to the chromatin proteins using formaldehyde as a crosslinking agent and sheared to small pieces. The free DNA is afterwards enriched using phenol:chloroform extraction. The ratio of free DNA is determined by quantitative polymerase chain reaction (qPCR) or DNA sequencing (DNA-seq) compared to a control sample representing total DNA. The regions with a looser chromatin structure are enriched in the free DNA sample, thus allowing the identification of genomic regions with lower chromatin compaction.

Structural characterization of Set1 RNA recognition motifs and their role in histone H3 lysine 4 methylation.

The yeast Set1 histone H3 lysine 4 (H3K4) methyltransferase contains, in addition to its catalytic SET domain, a conserved RNA recognition motif (RRM1). We present here the crystal structure and the secondary structure assignment in solution of the Set1 RRM1. Although RRM1 has the expected betaalphabetabetaalphabeta RRM-fold, it lacks the typical RNA-binding features of these modules. RRM1 is not able to bind RNA by itself in vitro, but a construct combining RRM1 with a newly identified downstream RRM2 specifically binds RNA. In vivo, H3K4 methylation is not affected by a point mutation in RRM2 that preserves Set1 stability but affects RNA binding in vitro. In contrast mutating RRM1 destabilizes Set1 and leads to an increase of dimethylation of H3K4 at the 5'-coding region of active genes at the expense of trimethylation, whereas both, dimethylation decreases at the 3'-coding region. Taken together, our results suggest that Set1 RRMs bind RNA, but Set1 RNA-binding activity is not linked to H3K4 methylation.

Testing the Effects of SIAH Ubiquitin E3 Ligases on Lysine Acetyl Transferases.

The family of seven-in-absentia (SIAH) ubiquitin E3 ligases functions in the control of numerous key signaling pathways. These enzymes belong to the RING (really interesting new gene) group of E3 ligases and mediate the attachment of ubiquitin chains to substrates, which then leads to their proteasomal degradation. Here, we describe a protocol that allows measuring SIAH-mediated ubiquitination and degradation of its client proteins as exemplified by acetyl transferases using simple overexpression experiments. The impact of SIAH expression on the relative amounts of target proteins and their mRNAs can be quantified by Western blotting and quantitative PCR (qPCR) as described here.

The CCR4-NOT complex contributes to repression of Major Histocompatibility Complex class II transcription.

The multi-subunit CCR4 (carbon catabolite repressor 4)-NOT (Negative on TATA) complex serves as a central coordinator of all different steps of eukaryotic gene expression. Here we performed a systematic and comparative analysis of cells where the CCR4-NOT subunits CNOT1, CNOT2 or CNOT3 were individually downregulated using doxycycline-inducible shRNAs. Microarray experiments showed that downregulation of either CNOT subunit resulted in elevated expression of major histocompatibility complex class II (MHC II) genes which are found in a gene cluster on chromosome 6. Increased expression of MHC II genes after knock-down or knock-out of either CNOT subunit was seen in a variety of cell systems and also in naïve macrophages from CNOT3 conditional knock-out mice. CNOT2-mediated repression of MHC II genes occurred also in the absence of the master regulator class II transactivator (CIITA) and did not cause detectable changes of the chromatin structure at the chromosomal MHC II locus. CNOT2 downregulation resulted in an increased de novo transcription of mRNAs whereas tethering of CNOT2 to a regulatory region governing MHC II expression resulted in diminished transcription. These results expand the known repertoire of CCR4-NOT members for immune regulation and identify CNOT proteins as a novel group of corepressors restricting class II expression.