The role of the epidermis enhancer element in positive and negative transcriptional regulation of ebony in Drosophila melanogaster

The spatiotemporal regulation of gene expression is essential to ensure robust phenotypic outcomes. Pigmentation patterns in Drosophila are determined by pigments biosynthesized in the developing epidermis and the cis-regulatory elements (CREs) of the genes involved in this process are well-characterized. Here we report that the known primary epidermal enhancer (priEE) is dispensable for the transcriptional activation of ebony (involved in light-colored pigment synthesis) in the developing epidermis of D. melanogaster. The evidence was obtained by introducing an approximately 1 kbp deletion at the priEE by genome editing. The effect of the priEE deletion on pigmentation and on the endogenous expression pattern of a mCherry-fused ebony allele was examined in the abdomen. The expression levels of the mCherry-fused ebony in the priEE-deleted strains were slightly higher than that of the control strain, indicating that the sequences outside the priEE have an ability to drive an expression of this gene in the epidermis. Interestingly, the priEE deletion resulted in a derepression of this gene in the dorsal midline of the abdominal tergites, where dark pigmentation is present in the wild-type individuals. This indicated that the priEE fragment contains a silencer. Furthermore, the endogenous expression pattern of ebony in the two additional strains with partially deleted priEE revealed that the silencer resides within a 351-bp fragment in the 5' portion of the priEE. These results demonstrated that deletion assays combined with reporter assays are highly effective in detecting the presence of positively and negatively regulating sequences within and outside the focal CREs.

Tcf1 is essential for initiation of oncogenic Notch1-driven chromatin topology in T-ALL

NOTCH1 is a well-established lineage specifier for T cells and amongst the most frequently mutated genes throughout all subclasses of T cell acute lymphoblastic leukemia (T-ALL). How oncogenic NOTCH1 signaling launches a leukemia-prone chromatin landscape during T-ALL initiation is unknown. Here we demonstrate an essential role for the high-mobility-group transcription factor Tcf1 in orchestrating chromatin accessibility and topology allowing aberrant Notch1 signaling to convey its oncogenic function. Although essential, Tcf1 is not sufficient to initiate leukemia. The formation of a leukemia-prone epigenetic landscape at the distal Notch1-regulated Myc enhancer, which is fundamental to this disease, is Tcf1-dependent and occurs within the earliest progenitor stage even before cells adopt a T lymphocyte or leukemic fate. Moreover, we discovered a unique evolutionarily conserved Tcf1-regulated enhancer element in the distal Myc-enhancer, which is important for the transition of pre-leukemic cells to full-blown disease.

An evolutionarily conserved cis-regulatory element of Nkx3.2 contributes to early jaw joint morphology in zebrafish

The acquisition of movable jaws was a major event during vertebrate evolution. The role of NK3 homeobox 2 (Nkx3.2) transcription factor in patterning the primary jaw joint of gnathostomes (jawed vertebrates) is well known, however knowledge about its regulatory mechanism is lacking. In this study, we report a proximal enhancer element of Nkx3.2 that is deeply conserved in gnathostomes but undetectable in the jawless hagfish. This enhancer is active in the developing jaw joint region of the zebrafish Danio rerio, and was thus designated as jaw joint regulatory sequence 1 (JRS1). We further show that JRS1 enhancer sequences from a range of gnathostome species, including a chondrichthyan and mammals, have the same activity in the jaw joint as the native zebrafish enhancer, indicating a high degree of functional conservation despite the divergence of cartilaginous and bony fish lineages or the transition of the primary jaw joint into the middle ear of mammals. Finally, we show that deletion of JRS1 from the zebrafish genome using CRISPR/Cas9 leads to a transient jaw joint deformation and partial fusion. Emergence of this Nkx3.2 enhancer in early gnathostomes may have contributed to the origin and shaping of the articulating surfaces of vertebrate jaws.

Interchromosomal interaction of homologous Stat92E alleles regulates transcriptional switch during stem-cell differentiation

The strength of pairing of homologous chromosomes differs in a locus-specific manner and is correlated to gene expression states. However, the functional impact of homolog pairing on local transcriptional activity is still unclear. Drosophila male germline stem cells (GSCs) constantly divide asymmetrically to produce one GSC and one differentiating gonialblast (GB). The GB then enters the differentiation program in which stem cell specific genes are quickly downregulated. Here we demonstrate that a change in local pairing state of Stat92E locus is required for the downregulation of the Stat92E gene during differentiation. Using OligoPaint fluorescent in situ hybridization (FISH), we show that the interaction between homologous loci of Stat92E is always tight in GSCs and immediately loosened in GBs. When one of the Stat92E locus was absent or relocated to another chromosome, Stat92E did not pair and failed to downregulate, suggesting that the pairing is required for switching of transcriptional activity. The defect in downregulation of Stat92E was also observed upon knockdown of global pairing or anti-pairing factors. Moreover, the Stat92E enhancer element, but not cis-transcription, is required for the change in pairing state, indicating that it is not a consequence of transcriptional changes. GSCs are known to inherit pre-existing histones H3 and H4, while newly synthesized histones are distributed in GBs. When this histone inheritance was compromised, the change in Stat92E pairing did not occur, suggesting that it is an intrinsically programmed process during asymmetric stem cell division. We propose that the change of local pairing state may be a common process to reprogram gene activity during cell-differentiation.

SATB1 Regulates Chromatin Organization and HSP70 Expression in Early Erythropoiesis and Is Downregulated in Models of Diamond Blackfan Anemia

Diamond Blackfan Anemia (DBA) is a rare genetic disease predominantly caused by mutations carried within one of at least 20 ribosomal genes. DBA is characterized by red blood cell aplasia and normal myeloid and megakaryocyte progenitors, indicating that early uncommitted progenitors are relatively unaffected by the mutations. In DBA, the formation of BFU-E colonies and subsequent erythroblasts are severely restricted and indicate a defect in one of the earliest stages of erythroid expansion. To identify critical molecular mechanisms that may regulate early erythropoiesis, we used shRNAs against the ribosomal protein RPS19 (the most commonly mutated gene in DBA) in cord blood derived CD34+ hematopoietic stem and progenitor cells (HSPCs) and performed bulk RNA-seq. After 3 days in an erythroid culture media, the transcriptomes in CD71+ erythroid progenitors were examined. We found that the special AT binding protein 1 (SATB1) was downregulated in RPS19-insufficient HSPCs compared to healthy cord blood HSPCs. SATB1 is modestly expressed in hematopoietic stem cells but is induced during lymphoid expansion and has been previously reported to suppress myeloid/erythroid progenitor (MEP) expansion. Our results showed that maintaining SATB1 expression is required for optimal expansion of MEP progenitors and that the premature loss of SATB1 in DBA contributes to the anemia phenotype. SATB1 binds to 3 specific regions upstream of the 5'UTR of the HSP70 genes and induces the formation of 2 chromatin loops. An enhancer element associates with the proximal promoters of the two HSP70 genes and facilitates the induction of HSP70. In DBA, HSP70 is not induced and contributes to DBA pathogenesis. HSPA1A is induced 4.3-fold while HSPA1B is induced 3.1-fold. Increased expression of the master erythroid transcription factor GATA1 during erythropoiesis occurs in two phases. The first induction precedes a more dramatic induction that accompanies later stages of erythroid differentiation. The absence of SATB1 or HSP70 reduced the earlier GATA1 induction that accompany MEP expansion by 46.1% and 49.3% respectively. The number of MEPs in SATB1 knockdown HSPCs was reduced, resulting in a 24.5% reduction in CD235+ erythroid and 20.8% reduction in CD41+ megakaryocytes. While SATB1-independent effects of RPS19-insufficiency contribute more significantly to erythroid defects in DBA, we have uncovered that SATB1 contributes to regulation of the earliest stages of erythropoiesis by facilitating the induction of HSP70 and subsequent stabilization of an early induction of GATA1. Disclosures No relevant conflicts of interest to declare.

Soluble Urokinase Plasminogen Activator Receptor: Genetic Variation and Cardiovascular Disease Risk in Blacks

Background: suPAR (Soluble urokinase plasminogen activator receptor) has emerged as an important biomarker of coagulation, inflammation, and cardiovascular disease (CVD) risk. The contribution of suPAR to CVD risk and its genetic influence in the Black population have not been evaluated. Methods: We measured suPAR in 3492 Blacks from the prospective, community-based JHS (Jackson Heart Study). Cross-sectional associations of suPAR with lifestyle and CVD risk factors were assessed, whole-genome sequence data were used to evaluate genetic associations of suPAR, and relationships of suPAR with incident CVD outcomes and overall mortality were estimated over follow-up. Results: In Cox models adjusted for traditional CVD risk factors, estimated glomerular filtration rate, and CRP (C-reactive protein), each 1-SD higher suPAR was associated with a 21% to 31% increased risk of incident coronary heart disease, heart failure, stroke, and mortality. In the genome-wide association study, 2 missense (rs399145 encoding p.Thr86Ala, rs4760 encoding p.Phe272Leu) and 2 noncoding regulatory variants (rs73935023 within an enhancer element and rs4251805 within the promoter) of PLAUR on chromosome 19 were each independently associated with suPAR and together explained 14% of suPAR phenotypic variation. The allele frequencies of each of the four suPAR-associated genetic variants differ considerably across African and European populations. We further show that PLAUR rs73935023 can alter transcriptional activity in vitro. We did not find any association between genetically determined suPAR and CVD in JHS or a larger electronic medical record-based analyses of Blacks or Whites. Conclusions: Our results demonstrate the importance of ancestry-differentiated genetic variation on suPAR levels and indicate suPAR is a CVD biomarker in Black adults.

SNP-mediated binding of TBX1 to the enhancer element of IL-10 reduces the risk of Behcet’s disease

Aims: The genetic association between Behcet’s disease susceptibility and IL-10 has been confirmed in multiple cohorts, but the underlying mechanism of this association remains unclear. Materials & methods: We combined public resources and laboratory experiments (electrophoretic mobility shift assays, chromatin immunoprecipitation, luciferase reporter gene and CRISPR/Cas9 genome editing) to analyze transcription factor binding and enhancer activity controlling IL-10 expression. Results & conclusion: The T allele of noncoding rs3024490 within super-enhancer elements is able to specifically bind TBX1 and, in turn, promotes the enhancer activity and increased expression of IL-10. However, a relative deficiency in TBX1 in Behcet’s disease patients leads to the low expression of IL-10 and increased risk of developing Behcet’s disease.

RelB Upregulates PD-L1 and Exacerbates Prostate Cancer Immune Evasion

Background Interaction between programmed death receptor (PD-1) and its ligand (PD-L1) is essential for suppressing activated T-lymphocytes. However, the precise mechanisms underlying PD-L1 overexpression in tumors remain to be fully elucidated. Here, we describe that RelB participates in immune evasion of prostate cancer (PCa) via cis/trans transcriptional upregulation of PD-L1.MethodsBased on transcriptome results, RelB was manipulated in multiple human and murine PCa cell lines. The activated CD4+ and CD8+ T cells were co-cultured with PCa cells with different levels of RelB to examine the effect of tumorous RelB on T-cell immunity. Male mice were injected with murine PCa cells to validate the effect of RelB on the PD-1/PD-L1-mediated immune checkpoint using both tumor growing and metastatic experimental models.Results PD-L1 uniquely expresses at a high level in PCa with high constitutive RelB and correlates to the patients’ Gleason scores. Indeed, the ligh level of PD-L1 is associated with RelB nuclear translocatiob in AR-negative aggressive PCa cells. The silence of RelB in advanced PCa cells resulted in reducing PD-L1 expression and enhancing the susceptibility of PCa cells to the T-cell immune response in vitro and in vivo. Mechanistically, a proximal NF-kB enhancer element was identified in the core promoter region of the human CD274 gene, which is responsible for RelB-mediated PD-L1 transcriptional activation. The finding provides an informative insight into immune checkpoint blockade by administering RelB within the tumor microenvironment.ConclusionsThis study deciphers the molecular mechanism by which tumorous RelB contributes to immune evasion by inhibiting T-cell immunity via the amplification of PD-L1/PD-1-mediated immune checkpoint.

Short regulatory DNA sequences to target brain endothelial cells for gene therapy

Gene vectors targeting CNS endothelial cells allow to manipulate the blood-brain barrier and to correct genetic defects in the CNS. Because vectors based on the adeno-associated virus (AAV) have a limited capacity, it is essential that the DNA sequence controlling gene expression is short. In addition, it must be specific for endothelial cells to avoid off-target effects. To develop improved regulatory sequences with selectivity for brain endothelial cells, we tested the transcriptional activity of truncated promoters of eleven (brain) endothelial-specific genes in combination with short regulatory elements, i.e., the woodchuck post-transcriptional regulatory element (W), the CMV enhancer element (C), and a fragment of the first intron of the Tie2 gene (S), by transfecting brain endothelial cells of three species. Four combinations of regulatory elements and short promoters ( Cdh5, Ocln, Slc2a1, and Slco1c1) progressed through this in-vitro pipeline displaying suitable activity. When tested in mice, the regulatory sequences C- Ocln-W and C- Slc2a1-S-W enabled a stronger and more specific gene expression in brain endothelial cells than the frequently used CAG promoter. In summary, the new regulatory elements efficiently control gene expression in brain endothelial cells and may help to specifically target the blood-brain barrier with gene therapy vectors.