scholarly journals Insight into GATA1 transcriptional activity through interrogation of cis elements disrupted in human erythroid disorders

2016 ◽  
Vol 113 (16) ◽  
pp. 4434-4439 ◽  
Author(s):  
Aoi Wakabayashi ◽  
Jacob C. Ulirsch ◽  
Leif S. Ludwig ◽  
Claudia Fiorini ◽  
Makiko Yasuda ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Transcriptional Activity ◽  
Regulatory Elements ◽  
Binding Motif ◽  
Targeted Disruption ◽  
Altered Gene Expression ◽  
Whole Exome ◽  
Altered Gene ◽  
Insight Into

Whole-exome sequencing has been incredibly successful in identifying causal genetic variants and has revealed a number of novel genes associated with blood and other diseases. One limitation of this approach is that it overlooks mutations in noncoding regulatory elements. Furthermore, the mechanisms by which mutations in transcriptional cis-regulatory elements result in disease remain poorly understood. Here we used CRISPR/Cas9 genome editing to interrogate three such elements harboring mutations in human erythroid disorders, which in all cases are predicted to disrupt a canonical binding motif for the hematopoietic transcription factor GATA1. Deletions of as few as two to four nucleotides resulted in a substantial decrease (>80%) in target gene expression. Isolated deletions of the canonical GATA1 binding motif completely abrogated binding of the cofactor TAL1, which binds to a separate motif. Having verified the functionality of these three GATA1 motifs, we demonstrate strong evolutionary conservation of GATA1 motifs in regulatory elements proximal to other genes implicated in erythroid disorders, and show that targeted disruption of such elements results in altered gene expression. By modeling transcription factor binding patterns, we show that multiple transcription factors are associated with erythroid gene expression, and have created predictive maps modeling putative disruptions of their binding sites at key regulatory elements. Our study provides insight into GATA1 transcriptional activity and may prove a useful resource for investigating the pathogenicity of noncoding variants in human erythroid disorders.

Abstract P323: HIV- and Subclinical Cardiovascular Disease-specific Transcriptomes in Nonclassical Monocytes: The Women’s Interagency HIV Study

Circulation ◽  
2020 ◽  
Vol 141 (Suppl_1) ◽  
Author(s):  
Juan Lin ◽  
David B Hanna ◽  
Qibin Qi ◽  
tao wang ◽  
Karin A Mueller ◽  
...  
Keyword(s):  
Gene Expression ◽  
Lipid Homeostasis ◽  
Checkpoint Inhibition ◽  
Protein Coding ◽  
Altered Gene Expression ◽  
Subclinical Cardiovascular Disease ◽  
Altered Gene ◽  
Transcriptomic Changes ◽  
Insight Into ◽  
Chronic Hiv Infection

Objectives: Nonclassical monocytes (NCM) have patrolling functions relevant to atherosclerosis. While NCM have low surface CXCR4 expression in people with concurrent HIV and CVD (Mueller Cardiovasc Res 2019), the extent of CVD-related gene expression and the pathways involved are unknown. We described the gene transcription signature of NCM to provide insight into potential mechanisms of HIV-associated CVD. Methods: We identified transcriptomic changes in circulating NCM among women with and without chronic HIV infection. CVD was defined by plaques found on B-mode carotid artery ultrasound. The study included 23 HIV - CVD - , 21 HIV + CVD - , 20 HIV - CVD + , and 21 HIV + CVD + women, with these four groups matched by age (median = 45), race (95% minority) and smoking (86% ever-smokers). Using cryopreserved cells, we flow-sorted NCM (CD14 dim CD16+) and deep-sequenced their mRNA (average depth >40 million reads) to identify differentially expressed genes (DEG) contrasting HIV alone, CVD alone, and concurrent HIV + CVD + groups, versus HIV - CVD - , based on FDR-adjusted P<0.05. Results: After filtering to genes with raw counts >10 in >60% of participants, 11,343 protein coding genes were analyzed. HIV alone was associated with 10 DEGs on NCM (Figure). Women affected by both HIV and CVD had 93 DEGs, only six of which were shared by the HIV alone DEG signal. CVD alone was associated only with upregulated CDK18, which was also identified as a DEG in the HIV + CVD + group. Conclusion: Concurrent HIV and CVD (HIV + CVD + ) is associated with altered gene expression in NCMs relative to HIV - CVD - , generating responses that involve interleukins (IL32, IL4R), immune checkpoint inhibition (LAG3), chemokines (CCL4, CCL5) and lipid homeostasis (ABCD2).

ChIP-on-Chip Analysis Identified HLXB9 as a Transcription Factor In Hematopoietic Cells and Alters the Expression of Genes Involved In Cell-Adhesion

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2486-2486
Author(s):  
Sarah Wildenhain ◽  
Christian Ruckert ◽  
Svenja Daschkey ◽  
Martin Dugas ◽  
Julia Hauer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Cell Adhesion ◽  
Hematopoietic Cells ◽  
Promoter Regions ◽  
Altered Gene Expression ◽  
Altered Gene ◽  
On Chip ◽  
Chip Analysis

Abstract Abstract 2486 Infants with t (7;12)/HLXB9-TEL positive Acute Myeloid Leukemia (AML) have an Event-Free Survival (EFS) of 0 % and are characterized by concomitant HLXB9 (MNX1) expression. However, the role of the homeobox protein HLXB9 on hematopoietic cell development remains unknown. Expression profiling of t (7;12) and t (11;X) positive leukemias revealed up-regulation of cell-cell interacting genes in t (7;12) positive leukemia (Wildenhain et al., 2010). Furthermore, no increased expression of HOX-Genes, like HOXA9 and MEIS1, could be observed in t (7;12) positive leukemia compared to t (11;X) positive leukemia. Based on the altered gene expression profile in t (7;12) positive leukemia we investigated the role of HLXB9 as a transcription factor in hematopoietic cells using ChIP-on-chip analysis and its impact on the cellular gene expression pattern using Affymetrix expression arrays. The myeloid cell line HL60 was stable transfected with a CMV-HLXB9 (HL60/HLXB9) expression vector or an empty vector control (HL60/control). Microarray analysis was performed using “Human Gene 1.0 ST Arrays” (Affymetrix) and data from the HL60/HLXB9 cells were normalized to HL60/control cells. ChIP-on-chip analysis was performed using the “SimpleChIP Enzymatic Chromatin IP Kit” (Cell Signaling Technologies). Hybridisation on “385K RefSeq Promoter arrays” and analysis of raw data were performed by NimbleGen using the NimbleScan software. Data were visualized with the SignalMap software. Altered expression analyses as well as enrichment of promoter regions were validated by quantitative RT-PCR. Expression analysis revealed 81 differentially expressed genes, whereof 63 were down-regulated indicating that HLXB9 acts as a transcriptional repressor, as characteristic for homeobox proteins. CLEC5A, normally expressed in mature myeloid cells, is the highest differentially repressed gene. Further, we identified several differentially expressed genes which interfere in cell-adhesion and/or angiogenesis (e.g. IL8, ZYX, SELL, SPP1, EMILIN2). Western blot analysis of nuclear extracts confirmed the translocation of HLXB9 into the nucleus. ChIP-on-chip analysis revealed binding of HLXB9 to several promoter regions, among them the promoters of ZYX and IL8. Binding of HLXB9 to those promoters results in a decreased gene expression.These data strengthens the hypothesis, that HLXB9 plays a major role in cell adhesion and/or cell interactions. Further we observed increased expression of the adhesion molecule CD11b, when culturing HL60/HLXB9 cells in All-Trans Retinoic Acid (ATRA) containing medium in contrast to HL60/control cells. In summary, this study shows that HLXB9 acts as a transcription factor in hematopoietic cells and has a repressive function on gene expression. HLXB9 target genes regulate cell-adhesion and angiogenesis. This study provides the first molecular results of HLXB9 function in hematopoietic cells and supports the previously published data showing the importance on altered gene expression of cell-cell interacting genes in the pathogenesis of t (7;12) positive leukemia. Disclosures: No relevant conflicts of interest to declare.

scholarly journals An essential role for MEF2C in the cortical response to loss of sleep

2020 ◽  
Author(s):  
Theresa E. Bjorness ◽  
Ashwinikumar Kulkarni ◽  
Volodymer Rybalchenko ◽  
Ayako Suzuki ◽  
Catherine Bridges ◽  
...  
Keyword(s):  
Gene Expression ◽  
Slow Wave ◽  
Transcriptional Activity ◽  
Slow Wave Sleep ◽  
Sleep Loss ◽  
Wave Activity ◽  
Altered Gene Expression ◽  
Mepsc Frequency ◽  
Differential Gene ◽  
Altered Gene

AbstractNeuronal activity and gene expression in response to the loss of sleep can provide a window into the enigma of sleep function. Sleep loss is associated with brain differential gene expression, an increase in pyramidal cell mEPSC frequency and amplitude, and a characteristic rebound and resolution of slow wave sleep-slow wave activity (SWS-SWA). However, the molecular mechanism(s) mediating the sleep loss response are not well understood. We show that sleep-loss regulates MEF2C phosphorylation, a key mechanism regulating MEF2C transcriptional activity, and that MEF2C function in postnatal excitatory forebrain neurons is required for the biological events in response to sleep loss. These include altered gene expression, the increase and recovery of synaptic strength, and the rebound and resolution of SWS-SWA, which implicate MEF2C as an essential regulator of sleep function.One Sentence SummaryMEF2C is critical to the response to sleep loss.

scholarly journals An essential role for MEF2C in the cortical response to loss of sleep in mice

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Theresa E Bjorness ◽  
Ashwinikumar Kulkarni ◽  
Volodymyr Rybalchenko ◽  
Ayako Suzuki ◽  
Catherine Bridges ◽  
...  
Keyword(s):  
Gene Expression ◽  
Slow Wave ◽  
Transcriptional Activity ◽  
Slow Wave Sleep ◽  
Sleep Loss ◽  
Wave Activity ◽  
Altered Gene Expression ◽  
Mepsc Frequency ◽  
Differential Gene ◽  
Altered Gene

Neuronal activity and gene expression in response to the loss of sleep can provide a window into the enigma of sleep function. Sleep loss is associated with brain differential gene expression, an increase in pyramidal cell mEPSC frequency and amplitude, and a characteristic rebound and resolution of slow wave sleep-slow wave activity (SWS-SWA). However, the molecular mechanism(s) mediating the sleep-loss response are not well understood. We show that sleep-loss regulates MEF2C phosphorylation, a key mechanism regulating MEF2C transcriptional activity, and that MEF2C function in postnatal excitatory forebrain neurons is required for the biological events in response to sleep loss in C57BL/6J mice. These include altered gene expression, the increase and recovery of synaptic strength, and the rebound and resolution of SWS-SWA, which implicate MEF2C as an essential regulator of sleep function.

scholarly journals Sequence determinants of human gene regulatory elements

2021 ◽  
Author(s):  
Biswajyoti Sahu ◽  
Tuomo Hartonen ◽  
Paivi Pihlajamaa ◽  
Bei Wei ◽  
Kashyap Dave ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Sequences ◽  
Transcriptional Activity ◽  
Cell Types ◽  
Regulatory Elements ◽  
Binding Motif ◽  
Atomic Unit ◽  
Gene Regulatory Elements ◽  
A Cell ◽  
Different Cell Types

DNA determines where and when genes are expressed, but the full set of sequence determinants that control gene expression is not known. To obtain a global and unbiased view of the relative importance of different sequence determinants in gene expression, we measured transcriptional activity of DNA sequences that are in aggregate ~100 times longer than the human genome in three different cell types. We show that enhancers can be classified to three main types: classical enhancers1, closed chromatin enhancers and chromatin-dependent enhancers, which act via different mechanisms and differ in motif content. Transcription factors (TFs) act generally in an additive manner with weak grammar, with classical enhancers increasing expression from promoters by a mechanism that does not involve specific TF-TF interactions. Few TFs are strongly active in a cell, with most activities similar between cell types. Chromatin-dependent enhancers are enriched in forkhead motifs, whereas classical enhancers contain motifs for TFs with strong transactivator domains such as ETS and bZIP; these motifs are also found at transcription start site (TSS)-proximal positions. However, some TFs, such as NRF1 only activate transcription when placed close to the TSS, and others such as YY1 display positional preference with respect to the TSS. TFs can thus be classified into four non-exclusive subtypes based on their transcriptional activity: chromatin opening, enhancing, promoting and TSS determining factors — consistent with the view that the binding motif is the only atomic unit of gene expression.

scholarly journals The C. elegans neural editome reveals an ADAR target mRNA required for proper chemotaxis

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Sarah N Deffit ◽  
Brian A Yee ◽  
Aidan C Manning ◽  
Suba Rajendren ◽  
Pranathi Vadlamani ◽  
...  
Keyword(s):  
Gene Expression ◽  
Caenorhabditis Elegans ◽  
Rna Editing ◽  
Regulatory Elements ◽  
Neural Cells ◽  
Neuronal Function ◽  
C Elegans ◽  
Altered Gene Expression ◽  
Altered Gene ◽  
Editing Enzyme

ADAR proteins alter gene expression both by catalyzing adenosine (A) to inosine (I) RNA editing and binding to regulatory elements in target RNAs. Loss of ADARs affects neuronal function in all animals studied to date. Caenorhabditis elegans lacking ADARs exhibit reduced chemotaxis, but the targets responsible for this phenotype remain unknown. To identify critical neural ADAR targets in C. elegans, we performed an unbiased assessment of the effects of ADR-2, the only A-to-I editing enzyme in C. elegans, on the neural transcriptome. Development and implementation of publicly available software, SAILOR, identified 7361 A-to-I editing events across the neural transcriptome. Intersecting the neural editome with adr-2 associated gene expression changes, revealed an edited mRNA, clec-41, whose neural expression is dependent on deamination. Restoring clec-41 expression in adr-2 deficient neural cells rescued the chemotaxis defect, providing the first evidence that neuronal phenotypes of ADAR mutants can be caused by altered gene expression.

scholarly journals Landscape of tissue-specific RNA Editome provides insight into co-regulated and altered gene expression in pigs (Sus-scrofa)

RNA Biology ◽  
2021 ◽  
pp. 1-12
Author(s):  
A.ADETULA ET AL. Abiola Adetula ◽  
Xinhao Fan ◽  
Yongsheng Zhang ◽  
Yilong Yao ◽  
Junyu Yan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Sus Scrofa ◽  
Tissue Specific ◽  
Altered Gene Expression ◽  
Altered Gene ◽  
Insight Into

Molecular and Microscopic Analysis of Altered Gene Expression in Transgenic and Gene Targeted Mice

1996 ◽  
Vol 54 ◽  
pp. 12-13
Author(s):  
W. K. Jones ◽  
J. Robbins
Keyword(s):  
Gene Expression ◽  
Pulmonary Veins ◽  
Microscopic Analysis ◽  
Mouse Tissue ◽  
Adult Heart ◽  
Heavy Chains ◽  
Altered Gene Expression ◽  
Altered Gene ◽  
Pulmonary Myocardium

Two myosin heavy chains (MyHC) are expressed in the mammalian heart and are differentially regulated during development. In the mouse, the α-MyHC is expressed constitutively in the atrium. At birth, the β-MyHC is downregulated and replaced by the α-MyHC, which is the sole cardiac MyHC isoform in the adult heart. We have employed transgenic and gene-targeting methodologies to study the regulation of cardiac MyHC gene expression and the functional and developmental consequences of altered α-MyHC expression in the mouse.We previously characterized an α-MyHC promoter capable of driving tissue-specific and developmentally correct expression of a CAT (chloramphenicol acetyltransferase) marker in the mouse. Tissue surveys detected a small amount of CAT activity in the lung (Fig. 1a). The results of in situ hybridization analyses indicated that the pattern of CAT transcript in the adult heart (Fig. 1b, top panel) is the same as that of α-MyHC (Fig. 1b, lower panel). The α-MyHC gene is expressed in a layer of cardiac muscle (pulmonary myocardium) associated with the pulmonary veins (Fig. 1c). These studies extend our understanding of α-MyHC expression and delimit a third cardiac compartment.

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