A Two-Color Haploid Genetic Screen Identifies Novel Host Factors Involved in HIV-1 Latency

To identify novel host factors as putative targets to reverse HIV-1 latency, we performed an insertional mutagenesis genetic screen in a latent HIV-1 infected pseudohaploid KBM7 cell line (Hap-Lat). Following mutagenesis, insertions were mapped to the genome, and bioinformatic analysis resulted in the identification of 69 candidate host genes involved in maintaining HIV-1 latency. A select set of candidate genes was functionally validated using short hairpin RNA (shRNA)-mediated depletion in latent HIV-1 infected J-Lat A2 and 11.1 T cell lines. We confirmed ADK, CHD9, CMSS1, EVI2B, EXOSC8, FAM19A, GRIK5, IRF2BP2, NF1, and USP15 as novel host factors involved in the maintenance of HIV-1 latency. Chromatin immunoprecipitation assays indicated that CHD9, a chromodomain helicase DNA-binding protein, maintains HIV-1 latency via direct association with the HIV-1 5′ long terminal repeat (LTR), and its depletion results in increased histone acetylation at the HIV-1 promoter, concomitant with HIV-1 latency reversal. FDA-approved inhibitors 5-iodotubercidin, trametinib, and topiramate, targeting ADK, NF1, and GRIK5, respectively, were characterized for their latency reversal potential. While 5-iodotubercidin exhibited significant cytotoxicity in both J-Lat and primary CD4(+) T cells, trametinib reversed latency in J-Lat cells but not in latent HIV-1 infected primary CD4(+) T cells. Importantly, topiramate reversed latency in cell line models, in latently infected primary CD4(+) T cells, and crucially in CD4(+) T cells from three people living with HIV-1 (PLWH) under suppressive antiretroviral therapy, without inducing T cell activation or significant toxicity. Thus, using an adaptation of a haploid forward genetic screen, we identified novel and druggable host factors contributing to HIV-1 latency

The transcription factor BCL-6 controls early development of innate-like T cells

Innate T cells, including invariant natural killer T (iNKT) and mucosal-associated innate T (MAIT) cells, are a heterogeneous T lymphocyte population with effector properties preprogrammed during their thymic differentiation. How this program is initiated is currently unclear. Here, we show that the transcription factor BCL-6 was transiently expressed in iNKT cells upon exit from positive selection and was required for their proper development beyond stage 0. Notably, development of MAIT cells was also impaired in the absence of Bcl6. BCL-6-deficient iNKT cells had reduced expression of genes that were associated with the innate T cell lineage, including Zbtb16, which encodes PLZF, and PLZF-targeted genes. BCL-6 contributed to a chromatin accessibility landscape that was permissive for the expression of development-related genes and inhibitory for genes associated with naive T cell programs. Our results revealed new functions for BCL-6 and illuminated how this transcription factor controls early iNKT cell development

Catchet-MS identifies IKZF1-targeting thalidomide analogues as novel HIV-1 latency reversal agents

A major pharmacological strategy toward HIV cure aims to reverse latency in infected cells as a first step leading to their elimination. While the unbiased identification of molecular targets physically associated with the latent HIV-1 provirus would be highly valuable to unravel the molecular determinants of HIV-1 transcriptional repression and latency reversal, due to technical limitations, this has been challenging. Here we use a dCas9 targeted chromatin and histone enrichment strategy coupled to mass spectrometry (Catchet-MS) to probe the differential protein composition of the latent and activated HIV-1 5′LTR. Catchet-MS identified known and novel latent 5′LTR-associated host factors. Among these, IKZF1 is a novel HIV-1 transcriptional repressor, required for Polycomb Repressive Complex 2 recruitment to the LTR. We find the clinically advanced thalidomide analogue iberdomide, and the FDA approved analogues lenalidomide and pomalidomide, to be novel LRAs. We demonstrate that, by targeting IKZF1 for degradation, these compounds reverse HIV-1 latency in CD4+ T-cells isolated from virally suppressed people living with HIV-1 and that they are able to synergize with other known LRAs

HDAC7 is a major contributor in the pathogenesis of infant t(4;11) proB acute lymphoblastic leukemia

This paper was funded by grants to MP by the Spanish Ministry of Science, Innovation and Universities (SAF2017-87990-R and EUR2019-103835) and elaborated at the Josep Carreras Leukaemia Research Institute (IJC, Badalona, Barcelona) and IDIBELL Research Institute (L’Hospitalet de Llobregat, Barcelona). OdB is funded by a Juan de la Cierva—Formación fellowship from the Spanish Ministry of Science, Innovation and Universities (FJCI-2017-32430). AM is funded by the Spanish Ministry of Science, Innovation and Universities, which is part of the Agencia Estatal de Investigación (AEI), through grant PRE2018-083183 (cofunded by the European Social Fund). Work in PM and CB’s lab was supported by the European Research Council (CoG-2014646903), the Spanish Ministry of Economy and Competitiveness (SAF-2016-80481-R), Uno entre Cien Mil Foundation, the Leo Messi Foundation, the Asociación Española Contra el Cáncer (AECC-CI-2015), and the ISCIII/FEDER (PI17/01028).

Efficient Double Fragmentation ChIP-seq Provides Nucleotide Resolution Protein-DNA Binding Profiles

Immunoprecipitated crosslinked protein-DNA fragments typically range in size from several hundred to several thousand base pairs, with a significant part of chromatin being much longer than the optimal length for next-generation sequencing (NGS) procedures. Because these larger fragments may be non-random and represent relevant biology that may otherwise be missed, but also because they represent a significant fraction of the immunoprecipitated material, we designed a double-fragmentation ChIP-seq procedure. After conventional crosslinking and immunoprecipitation, chromatin is de-crosslinked and sheared a second time to concentrate fragments in the optimal size range for NGS. Besides the benefits of increased chromatin yields, the procedure also eliminates a laborious size-selection step. We show that the double-fragmentation ChIP-seq approach allows for the generation of biologically relevant genome-wide protein-DNA binding profiles from sub-nanogram amounts of TCF7L2/TCF4, TBP and H3K4me3 immunoprecipitated material. Although optimized for the AB/SOLiD platform, the same approach may be applied to other platforms

The Leukemia-Associated Mllt10/Af10-Dot1l Are Tcf4/β-Catenin Coactivators Essential for Intestinal Homeostasis

The leukemia-associated Mllt10/Af10 and its partner the histone methyltransferase Dot1l are identified as Tcf4/β-catenin co-activators and shown to be essential for Wnt-driven endogenous gene expression, intestinal development and homeostasis

Голодомор 1932 –– 1933 рр. в Україні як геноцид

Given its fundamental role in development and cancer, the Wnt-beta-catenin signaling pathway is tightly controlled at multiple levels. RING finger protein 43 (RNF43) is an E3 ubiquitin ligase originally found in stem cells and proposed to inhibit Wnt signaling by interacting with the Wnt receptors of the Frizzled family. We detected endogenous RNF43 in the nucleus of human intestinal crypt and colon cancer cells. We found that RNF43 physically interacted with T cell factor 4 (TCF4) in cells and tethered TCF4 to the nuclear membrane, thus silencing TCF4 transcriptional activity even in the presence of constitutively active mutants of beta-catenin. This inhibitory mechanism was disrupted by the expression of RNF43 bearing mutations found in human gastrointestinal tumors, and transactivation of the Wnt pathway was observed in various cells and in Xenopus embryos when the RING domain of RNF43 was mutated. Our findings indicate that RNF43 inhibits the Wnt pathway downstream of oncogenic mutations that activate the pathway. Mimicking or enhancing this inhibitory activity of RNF43 may be useful to treat cancers arising from aberrant activation of the Wnt pathwa

Study of the transcriptional regulation of hepatpcyte nuclear factor 4α

The work presented in the following pages focused on the elucidation of the mechanisms involved in the transcriptional regulation of the gene of hepatic nuclear factor 4α (HNF4α), a transcription factor which is expressed in a variety of tissues, such as the liver, the pancreas, the intestine and the kidney and which plays a significant role in the establishment and maintenance of the differentiated phenotype, in the organization of the specialized functions of those tissues. This is achieved through its participation in a regulatory network of transcription factors, the interplay and the interaction with relevant target genes of which determines the balanced expression of those gene products that are responsible for the function of the tissue. This work aimed at the understanding of the factors and mechanisms through which the expression of human HNF4α is regulated. To this end, as is presented in the first part of the work, 12.1 kb of upstream sequences of the gene were cloned and analyzed. Major sites of DNaseI hypersensitivity were identified both in the proximal promoter region and the first intron of the gene and in the upstream region, -6.6, -8.0 and -8.8 kb 5’ of the transcription start site. Transient transfection experiments using reporter genes under the control of progressive 5’ deletions of the regulatory region demonstrated that the region of the proximal promoter was sufficient for the induction of high levels of transcription in hepatic cells. DNaseI footprinting analysis and electrophoretic mobility shift assays revealed binding sites for the transcription factors HNF1α and β, Sp1, GATA6 and HNF6 in this region. Synergy between HNF1α and HNF6 and HNF1β and GATA6 produced high levels of transcription from the promoter, indicating that there are at least two alternative mechanisms for the transcriptional activation of the HNF4α gene. Chromatin immunoprecipitation experiments demonstrated that in differentiated hepatic cells it is the transcription factos HNF1α, HNF6, Sp1 and COUPTFII that interact with the promoter. The latter represses the promoter through a hormone response element that is not conserved in the homologous mouse region. This element was shown to respond to retinoic acid signaling, as incubation of cells with RA leads to the recruitment of the respective receptors to the promoter and to the upregulation of HNF4α expression levels. The second part of this work investigates the order of factor recruitment and chromatin modifications to the proximal promoter and an upstream enhancer of the human HNF4α gene, located -6.6 kb upstream of the transcription start site, around the initial activation of the gene during the differentiation of human enterocytes. These experiments demonstrated that two independent protein complexes formed in the two regulatory regions, which included transcription factors, acetyltransferases and chromatin remodelers at the enhancer and transcription factors, general transcription factors and RNA polymerase-II at the promoter. These events were followed by a unidirectional movement of the enhancer complex along the intermediate regions until it encountered the proximal promoter. This movement was accompanied by a spreading of histone hyperacetylation from the enhancer towards the promoter. The initiation of transcription from the gene coincided with the formation of a stable enhancer-promoter complex and the remodeling of the nucleosomes covering the transcription start site. These results gave a more complete picture of the events that take place in the regulatory regions of human HNF4α around the initial activation of the gene during a cellular differentiation model. They also provided experimental validation for a mechanism by which the communication between topologically remote regulatory regions is achieved through their physical contact and thus gave a mote complete picture of the complexity of the process of the transcriptional of at least one tissue-specific gene.Η παρούσα εργασία επικεντρώθηκε στη διασαφήνιση των μηχανισμών μεταγραφικής ενεργοποίησης του γονιδίου του ανθρώπινου ηπατικού πυρηνικού παράγοντα 4α (hepatic nuclear factor 4α, HNF4α). Ο HNF4α είναι ένας μεταγραφικός παράγοντας ο οποίος εκφράζεται σε μία πληθώρα ιστών, όπως το ήπαρ, το πάγκρεας, το έντερο και το νεφρό και παίζει σημαντικότατο ρόλο στη διατήρηση του διαφοροποιημένου φαινοτύπου, στην οργάνωση της επιτέλεσης, δηλαδή, των εξειδικευμένων λειτουργιών των ιστών αυτών. Αυτή τη λειτουργία την επιτυγχάνει μέσω της συμμετοχής του σε ένα ρυθμιστικό δίκτυο μεταγραφικών παραγόντων, των οποίων οι αλληλεπιδράσεις τόσο μεταξύ τους όσο και με τα γονίδια- στόχους τους καθορίζουν την ισορροπημένη έκφραση των γονιδιακών προϊόντων που είναι υπεύθυνα για την ομαλή λειτουργία του ιστού. Η εργασία αυτή στόχευε στην κατανόηση των παραγόντων και μηχανισμών με τους οποίους η ρυθμίζεται η έκφραση του ανθρώπινου HNF4α. Προς αυτό το σκοπό, όπως παρουσιάζεται στο πρώτο μέρος, κλωνοποιήθηκαν και αναλύθηκαν περίπου 12,1 kb αλληλουχιών 5’ του γονιδίου. Ταυτοποιήθηκαν κύριες θέσεις υπερευαισθησίας σε DNaseI τόσο στον εγγύς υποκινητή και στο πρώτο ιντρόνιο του γονιδίου, όσο και στην 5’ περιοχή, στις θέσεις -6,6, -8,0 και -8,8 kb 5’ του σημείου έναρξης της μεταγραφής. Πειράματα παροδικής διαμόλυνσης με γονίδιο αναφοράς υπό των έλεγχο προοδευτικών 5’ ελλείψεων της ρυθμιστικής περιοχής έδειξαν ότι η περιοχή του εγγύς υποκινητή ήταν ικανή να επάγει υψηλά επίπεδα μεταγραφής σε ηπατικά κύτταρα. Πειράματα ανάλυσης αποτυπωμάτων μεταγραφικών παραγόντων σε DNA (DNaseI footprinting) και μεταβολής της ηλεκτροφορητικής κινητικότητας του DNA (EMSA) αποκάλυψαν θέσεις πρόσδεσης για τους μεταγραφικούς παράγοντες HNF1α και β, Sp1, GATA6 και HNF6 σε αυτή την περιοχή. Η συνεργατική δράση των HNF1α και HNF6 και των HNF1β και GATA6 αντίστοιχα επήγαγε υψηλά επίπεδα μεταγραφής από τον υποκινητή, κάτι που έδειξε ότι υπάρχουν τουλάχιστον δύο εναλλακτικοί μηχανισμοί μεταγραφικής ενεργοποίησης του γονιδίου του HNF4α. Σε ότι αφορά την μεταγραφή του γονιδίου σε διαφοροποιημένα ηπατοκύτταρα, πειράματα ανοσοκατακρήμνισης χρωματίνης έδειξαν ότι, σε ανθρώπινη ηπατική κυτταρική σειρά, οι παράγοντες HNF1α, HNF6, Sp1 και COUPTFII αλληλεπιδρούν σταθερά με τον υποκινητή. Ο τελευταίος καταστέλλει τη δράση του υποκινητή μέσω ενός στοιχείου απόκρισης σε ορμόνες (hormone response element), το οποίο μάλιστα δεν υπάρχει στον ομόλογο υποκινητή του ποντικού. Το στοιχείο αυτό δείχθηκε ότι αποκρίνεται και σε ρετινοϊκό οξύ, καθώς επώαση κυττάρων με το τελευταίο οδηγεί στη στρατολόγηση των αντίστοιχων υποδοχέων στον υποκινητή και αύξηση των επιπέδων έκφρασης του HNF4α. Στο δεύτερο μέρος της εργασίας παρουσιάζεται η μελέτη της σειράς στρατολόγησης παραγόντων και των μεταβολών της χρωματίνης στον εγγύς υποκινητή και σε έναν 5’ ενισχυτή του HNF4α -6,6 kb από το σημείο έναρξης της μεταγραφής κατά την αρχική ενεργοποίηση του γονιδίου στη διάρκεια της διαφοροποίησης ανθρώπινων εντερικών κυττάρων. Τα πειράματα αυτά έδειξαν ότι στις δύο αυτές περιοχές σχηματίζονταν αρχικά ανεξάρτητα συσσωματώματα πρωτεϊνών, που περιελάμβαναν μεταγραφικούς παράγοντες, ακετυλοτρανσφεράσες και αναδιαμορφωτές της χρωματίνης στον ενισχυτή και μεταγραφικούς παράγοντες, γενικούς μεταγραφικούς παράγοντες και την RNA πολυμεράση-ΙΙ στον υποκινητή του γονιδίου. Τα γεγονότα αυτά ακολουθούσε μετακίνηση του συμπλόκου του ενισχυτή κατά μήκος των ενδιάμεσων περιοχών μέχρι τη συνάντησή του με τον υποκινητή. Η κίνηση αυτή συνοδευόταν από εξάπλωση της υπερακετυλίωσης των ιστονών από τον ενισχυτή αρχικά προς τον υποκινητή. Η έναρξη της μεταγραφής του γονιδίου συνέπιπτε με το σχηματισμό ενός σταθερού συσσωματώματος αποτελούμενου από τις πρωτεΐνες και των δύο περιοχών. Συνέπιπτε επίσης με την αναδιαμόρφωση του νουκλεοσώματος που κάλυπτε τη θέση έναρξης της μεταγραφής. Τα αποτελέσματα αυτά έδωσαν συνολικά μία πιο πλήρη εικόνα για τα γεγονότα που συμβαίνουν στις ρυθμιστικές περιοχές του HNF4α κατά την αρχική ενεργοποίηση του γονιδίου στη διάρκεια ενός μοντέλου κυτταρικής διαφοροποίησης. Παρείχαν επίσης πειραματική επιβεβαίωση για ένα μηχανισμό με τον οποίο επιτυγχάνεται η επικοινωνία μεταξύ τοπολογικά απομακρυσμένων ρυθμιστικών περιοχών μέσω φυσικής επαφής τους και έδωσαν έτσι μία πιο πλήρη εικόνα της πολυπλοκότητας της διαδικασίας της μεταγραφικής ενεργοποίησης ενός τουλάχιστον ιστο-ειδικού γονιδίου

Mitogen-Activated Protein Kinase-Mediated Disruption of Enhancer-Promoter Communication Inhibits Hepatocyte Nuclear Factor 4α Expression

Hepatocyte nuclear factor 4 (HNF-4) is a key member of the transcription factor network regulating hepatocyte differentiation and function. Activation of the HNF-4 gene involves physical interaction between a distant enhancer and the proximal promoter region, bound by distinct sets of transcription factors. Here we report that, upon mitogen-activated protein (MAP) kinase activation, HNF-4 expression is downregulated in human hepatoma cells. This effect is mediated by the loss of CEBPα expression. During MAP kinase signaling, the recruitment of HNF-3β and HNF-1α to the HNF-4 enhancer and RNA polymerase II to the proximal HNF-4 promoter was compromised. CBP, Brg1, and TFIIB were also dissociated from the HNF-4 regulatory regions, and the enhancer-promoter complex was disrupted. Interestingly, the extent of nucleosome acetylation did not decrease at either regulatory region, and HNF-6 and HNF-1α, as well as components of the TFIID, remained associated with the proximal promoter during the repressed state. The results point to an absolute requirement of enhancer-promoter communication for maintaining the active state of the HNF-4 gene and provide evidence for a molecular bookmarking mechanism, which may contribute to the prevention of permanent silencing of the locus during the repressed state

Crosstalk mechanisms between the WNT signaling pathway and long non-coding RNAs

The WNT/β-catenin signaling pathway controls a plethora of biological processes throughout animal development and adult life. Because of its fundamental role during animal lifespan, the WNT pathway is subject to strict positive and negative multi-layered regulation, while its aberrant activity causes a wide range of pathologies, including cancer. At present, despite the inroads into the molecules involved in WNT-mediated transcriptional responses, the fine-tuning of WNT pathway activity and the totality of its target genes have not been fully elucidated. Over the past few years, long non-coding RNAs (lncRNAs), RNA transcripts longer that 200nt that do not code for proteins, have emerged as significant transcriptional regulators. Recent studies show that lncRNAs can modulate WNT pathway outcome by affecting gene expression through diversified mechanisms, from the transcriptional to post-translational level. In this review, we selectively discuss those lncRNA-mediated mechanisms we believe the most important to WNT pathway modulation