Proximity of H2A.Z containing nucleosome to the transcription start site influences gene expression levels in the mammalian liver and brain

Control of gene expression in intestinal cells is poorly understood. Molecular mechanisms that regulate transcription of cellular genes are the foundation for understanding developmental and differentiation events. Mucin gene expression has been shown to be altered in many intestinal diseases and especially cancers of the gastrointestinal tract. Towards understanding the transcriptional regulation of a member of the 11p15.5 human mucin gene cluster, we have characterized 3.55 kb of the 5ʹ-flanking region of the human mucin gene MUC5B, including the promoter, the first two exons and the first intron. We report here the promoter activity of successively 5ʹ-truncated sections of 956 bases of this region by fusing it to the coding region of a luciferase reporter gene. The transcription start site was determined by primer-extension analysis. The region upstream of the transcription start site is characterized by the presence of a TATA box at bases -32/-26, DNA-binding elements for transcription factors c-Myc, N-Myc, Sp1 and nuclear factor ĸB as well as putative activator protein (AP)-1-, cAMP-response-element-binding protein (CREB)-, hepatocyte nuclear factor (HNF)-1-, HNF-3-, TGT3-, gut-enriched Krüppel factor (GKLF)-, thyroid transcription factor (TTF)-1- and glucocorticoid receptor element (GRE)-binding sites. Intron 1 of MUC5B was also characterized, it is 2511 nucleotides long and contains a DNA segment of 259 bp in which are clustered eight tandemly repeated GA boxes and a CACCC box that bind Sp1. AP-2α and GATA-1 nuclear factors were also shown to bind to their respective cognate elements in intron 1. In transfection studies the MUC5B promoter showed a cell-specific activity as it is very active in mucus-secreting LS174T cells, whereas it is inactive in Caco-2 enterocytes and HT-29 STD (standard) undifferentiated cells. Within the promoter, maximal transcription activity was found in a segment covering the first 223 bp upstream of the transcription start site. Finally, in co-transfection experiments a transactivating effect of Sp1 on to MUC5B promoter was seen in LS174T and Caco-2 cells.

Download Full-text

Alpha-thalassemia resulting from deletion of regulatory sequences far upstream of the alpha-globin structural genes

Blood ◽

10.1182/blood.v78.6.1589.bloodjournal7861589 ◽

1991 ◽

Vol 78 (6) ◽

pp. 1589-1595

Author(s):

L Romao ◽

L Osorio-Almeida ◽

DR Higgs ◽

J Lavinha ◽

SA Liebhaber

Keyword(s):

Gene Expression ◽

Transcription Start Site ◽

Globin Gene ◽

Regulatory Sequences ◽

Start Site ◽

Structural Genes ◽

Transcription Start ◽

Alpha Thalassemia ◽

Alpha Globin ◽

Globin Gene Expression

We describe an alpha-thalassemia determinant in which alpha-globin expression is silenced by a deletion located 27 kb 5′ to the transcription start site of the alpha 2-globin gene. This alpha- thalassemic determinant, (alpha alpha)MM, is a member of a newly described group of thalassemic mutations resulting from deletion of locus-controlling sequences critical to globin gene expression.

Download Full-text

A Chinese hamster transcription start site atlas that enables targeted editing of CHO cells

NAR Genomics and Bioinformatics ◽

10.1093/nargab/lqab061 ◽

2021 ◽

Vol 3 (3) ◽

Author(s):

Isaac Shamie ◽

Sascha H Duttke ◽

Karen J la Cour Karottki ◽

Claudia Z Han ◽

Anders H Hansen ◽

...

Keyword(s):

Gene Expression ◽

Transcription Start Site ◽

Cho Cells ◽

Genome Engineering ◽

Chinese Hamster ◽

Regulatory Elements ◽

Start Site ◽

Transcription Start ◽

Transcription Start Sites ◽

Silent Genes

Abstract Chinese hamster ovary (CHO) cells are widely used for producing biopharmaceuticals, and engineering gene expression in CHO is key to improving drug quality and affordability. However, engineering gene expression or activating silent genes requires accurate annotation of the underlying regulatory elements and transcription start sites (TSSs). Unfortunately, most TSSs in the published Chinese hamster genome sequence were computationally predicted and are frequently inaccurate. Here, we use nascent transcription start site sequencing methods to revise TSS annotations for 15 308 Chinese hamster genes and 3034 non-coding RNAs based on experimental data from CHO-K1 cells and 10 hamster tissues. We further capture tens of thousands of putative transcribed enhancer regions with this method. Our revised TSSs improves upon the RefSeq annotation by revealing core sequence features of gene regulation such as the TATA box and the Initiator and, as exemplified by targeting the glycosyltransferase gene Mgat3, facilitate activating silent genes by CRISPRa. Together, we envision our revised annotation and data will provide a rich resource for the CHO community, improve genome engineering efforts and aid comparative and evolutionary studies.

Download Full-text

Cooperation of E2F-p130 and Sp1-pRb Complexes in Repression of the Chinese Hamster dhfr Gene

Molecular and Cellular Biology ◽

10.1128/mcb.21.4.1121-1131.2001 ◽

2001 ◽

Vol 21 (4) ◽

pp. 1121-1131 ◽

Cited By ~ 54

Author(s):

Young-Chae Chang ◽

Sharon Illenye ◽

Nicholas H. Heintz

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Transcription Start Site ◽

Trichostatin A ◽

Chinese Hamster ◽

Serum Starvation ◽

Start Site ◽

Transcription Start ◽

Serum Stimulation ◽

Cell Extracts

ABSTRACT In mammalian cells reiterated binding sites for Sp1 and two overlapping and inverted E2F sites at the transcription start site regulate the dhfr promoter during the cell growth cycle. Here we have examined the contributions of the dhfr Sp1 and E2F sites in the repression of dhfr gene expression. In serum-starved cells or during serum stimulation, the Chinese hamsterdhfr gene was not derepressed by trichostatin A (TSA), an inhibitor of histone deacetylases (HDAC). Immunoprecipitation experiments showed that HDAC1 and hypophosphorylated retinoblastoma protein (pRb) are associated with Sp1 in serum-starved CHOC400 cells. In transfection experiments, reporter plasmids containing the reiterated dhfr Sp1 sites were stimulated 10-fold by TSA, while a promoter containing four dhfr E2F sites and a TATA box was responsive to E2F but was completely unaffected by TSA. HDAC1 did not coprecipitate with p130-E2F DNA binding complexes, the predominant E2F binding activity in cell extracts after serum starvation, suggesting that p130 imposes a TSA-insensitive state on thedhfr promoter. In support of this notion, recruitment of GAL4-p130 to a dihydrofolate reductase-GAL4 reporter rendered the promoter insensitive to TSA, while repression by GAL4-pRb was sensitive to TSA. Upon phosphorylation of pRb and p130 after serum stimulation, the Sp1-pRb and p130-E2F interactions were lost while the Sp1-HDAC1 interaction persisted into S phase. Together these studies suggest a dynamic model for the cooperation of pRb and p130 in repression ofdhfr gene expression during withdrawal from the cell cycle. We propose that, during initial phases of cell cycle withdrawal, the binding of dephosphorylated pRb to Sp1-HDAC1 complexes and complexes of E2F-1 -to -3 with DP results in transient, HDAC-dependent suppression of dhfr transcription. Upon withdrawal of cells into G0, recruitment of p130 to E2F-4–DP-1 complexes at the transcription start site results in a TSA-insensitive complex that cooperates with Sp1-HDAC-pRb complexes to stably repressdhfr promoter activity in quiescent cells.

Download Full-text

Characterization of the human proline/arginine-rich end leucine-rich repeat protein (PRELP) gene promoter and identification of a repressor element

Biochemical Journal ◽

10.1042/bj3360077 ◽

1998 ◽

Vol 336 (1) ◽

pp. 77-82 ◽

Cited By ~ 8

Author(s):

Judy GROVER ◽

Peter J. ROUGHLEY

Keyword(s):

Gene Expression ◽

Binding Site ◽

Transcription Start Site ◽

Gene Promoter ◽

Start Site ◽

Leucine Rich Repeat ◽

Transcription Start ◽

Repeat Protein ◽

Repressor Element ◽

Leucine Rich Repeat Protein

The 5´-flanking region of the human proline/arginine-rich end leucine-rich repeat protein (PRELP) gene has been characterized for both promoter and repressor activity by using a variety of reporter gene constructs and transient transfection into chondrocytes or fibroblasts. The human PRELP gene lacks a TATA box, and in its absence a Sp1-binding site residing 29 bp upstream of the transcription start site is essential for initiating gene expression. In contrast, an Ets-binding site residing 497 bp upstream of the transcription start site can lead to the repression of gene expression. The analysis of nuclear proteins by gel retardation studies with the repressor element identified a common protein, presumably an Ets family member, present in neonatal chondrocytes and skin fibroblasts that do not express the PRELP gene. The factor was not detected in nuclear protein preparations from adult chondrocytes in which the PRELP gene is expressed.

Download Full-text

Functional analysis of the human annexin I and VI gene promoters

Biochemical Journal ◽

10.1042/bj3320681 ◽

1998 ◽

Vol 332 (3) ◽

pp. 681-687 ◽

Cited By ~ 14

Author(s):

Shaun R. DONNELLY ◽

Stephen E. MOSS

Keyword(s):

Gene Expression ◽

Transcription Start Site ◽

Luciferase Reporter ◽

Gene Promoters ◽

Start Site ◽

Luciferase Reporter Gene ◽

Transcription Start ◽

A431 Cells ◽

Annexin I ◽

Annexin Vi

To gain insight into the molecular basis of annexin gene expression we have analysed the annexin I and VI gene promoters. A previously described 881 bp sequence immediately upstream of the annexin I transcription start site and a similar size fragment proximal to the annexin VI transcription start site both drove expression of the luciferase reporter gene in fibroblasts and epithelial cells. Neither promoter displayed any sensitivity to dexamethasone, suggesting that the putative glucocorticoid response element in the annexin I promoter is non-functional. Consistent with this, endogenous annexin I gene expression was unaffected by dexamethasone at the mRNA and protein levels in A431 cells. A series of 5´ deletions of the two promoters were examined to define the minimal active sequences. For annexin I this corresponded to a sequence approx. 150 bp upstream of the transcription start site that included CAAT and TATA boxes. Unexpectedly, the annexin VI promoter, which also contains CAAT and TATA boxes, was fully active in the absence of these elements, a 53 bp sequence between these boxes and the transcription start site having maximal activity. Electrophoretic mobility-shift assays with nuclear extracts from A431 and HeLa cells with probes corresponding to this region revealed an SP1-binding site. These results show that the annexin I and VI genes have individual modes of transcriptional regulation and that if either annexin I or annexin VI has an anti-inflammatory role, then this is in the absence of steroid-induced gene expression.

Download Full-text

Expression of the human oestrogen receptor-alpha gene is regulated by promoter F in MG-63 osteoblastic cells

Biochemical Journal ◽

10.1042/bj20021633 ◽

2003 ◽

Vol 372 (3) ◽

pp. 831-839 ◽

Cited By ~ 16

Author(s):

Elisabetta LAMBERTINI ◽

Letizia PENOLAZZI ◽

Silvia GIORDANO ◽

Laura DEL SENNO ◽

Roberta PIVA

Keyword(s):

Gene Expression ◽

Transcription Factors ◽

Transcription Start Site ◽

Thyroid Hormone Receptor ◽

Regulatory Elements ◽

Luciferase Reporter ◽

Start Site ◽

Mcf7 Cells ◽

Transcription Start ◽

Specific Element

(O)estrogen receptor-α (ERα), a hormone-dependent transcription factor belonging to the steroid/thyroid-hormone-receptor superfamily, plays an essential role in the development and maintenance of the skeleton. Here we report the analysis of an unexplored sequence inside the bone-specific distal promoter F (PF) with respect to the regulation of ERα gene expression in bone. This sequence, 785 bp in size, is localized upstream of the assigned transcription start site of exon F, at −117140 bp from the originally described transcription start site +1. It contains a TA reach box, a conventional CAAT box and potential regulatory elements for many transcription factors, including Cbfa1 [OSE2 (osteoblast-specific element) core binding factor], GATA-1 [(A/T)GATA(A/G) binding protein], Sox5 [sex-determining region Y (SRY)-type HMG bOX protein, belonging to a subfamily of DNA-binding proteins with an HMG domain], Sry, AP1 (activator protein 1) and CP2 (activator of γ-globin). It is able to strongly activate the luciferase reporter gene in MG-63 osteoblastic-like cells, but not in MCF7 breast-cancer cells. This is in agreement with different transcripts that we found in the two cell types. The footprinting and electrophoretic mobility-shift assays (EMSAs) showed that, inside the region analysed, there were some sequences that specifically reacted to nuclear proteins isolated from MG-63 cells. In particular, we identified two regions, named PFa and PFb, that do not present binding sites for known transcription factors and that are involved in a strong DNA–protein interaction in MG-63, but not in MCF7, cells. The analysis of three transcription factors (GATA-1, Sry and Sox) that might bind the identified footprinted areas suggested a possible indirect role of these proteins in the regulation of ERα gene expression in bone. These data provide evidence for different promoter usage of the ERα gene through the recruitment of tissue-specific transcription activators and co-regulators.

Download Full-text

The MuvB Complex Binds and Stabilizes Nucleosomes Downstream of the Transcription Start Site of Cell-Cycle Dependent Genes

10.1101/2021.06.29.450381 ◽

2021 ◽

Author(s):

Anushweta Asthana ◽

Parameshwaran Ramanan ◽

Alexander Hirschi ◽

Keelan Z Guiley ◽

Tilini U Wijeratne ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Transcription Start Site ◽

Nucleosome Occupancy ◽

Nucleosome Position ◽

Structural Data ◽

Start Site ◽

Transcription Start ◽

Cycle Dependent ◽

Differentiation And Proliferation

The chromatin architecture in promoters is thought to regulate gene expression, but it remains uncertain how most transcription factors (TFs) impact nucleosome position. The MuvB TF complex regulates cell-cycle dependent gene-expression and is critical for differentiation and proliferation during development and cancer. MuvB can both positively and negatively regulate expression, but the structure of MuvB and its biochemical function are poorly understood. Here we determine the overall architecture of MuvB assembly and the crystal structure of a subcomplex critical for MuvB function in gene repression. We find that the MuvB subunits LIN9 and LIN37 function as scaffolding proteins that arrange the other subunits LIN52, LIN54 and RBAP48 for TF, DNA, and histone binding, respectively. Biochemical and structural data demonstrate that MuvB binds nucleosomes through an interface that is distinct from LIN54-DNA consensus site recognition and that MuvB increases nucleosome occupancy in a reconstituted promoter. We find in arrested cells that MuvB primarily associates with a tightly positioned +1 nucleosome near the transcription start site (TSS) of MuvB-regulated genes. These results support a model that MuvB binds and stabilizes nucleosomes just downstream of the TSS on its target promoters to repress gene-expression.

Download Full-text

Genome-wide Chromatin Accessibility is Restricted by ANP32E

10.1101/2020.09.08.288241 ◽

2020 ◽

Author(s):

Kristin E. Murphy ◽

Fanju W. Meng ◽

Claire E. Makowski ◽

Patrick J. Murphy

Keyword(s):

Gene Expression ◽

Nucleosome Positioning ◽

Chromatin Accessibility ◽

Chromatin State ◽

Start Site ◽

Transcription Start ◽

Genome Wide ◽

Expression Potential ◽

Gene Expression Levels ◽

Hierarchical Manner

ABSTRACTGenome-wide chromatin state underlies gene expression potential and cellular function. Epigenetic features and nucleosome positioning contribute to the accessibility of DNA, but widespread regulators of chromatin state are largely unknown. Our study investigates how control of genomic H2A.Z localization by ANP32E contributes to chromatin state in mouse fibroblasts. We define H2A.Z as a universal chromatin accessibility factor, and demonstrate that through antagonism of H2A.Z, ANP32E restricts genome-wide DNA access. In the absence of ANP32E, H2A.Z accumulates at promoters in a hierarchical manner. H2A.Z initially localizes downstream of the transcription start site, and if H2A.Z is already present downstream, additional H2A.Z accumulates upstream. This hierarchical H2A.Z accumulation coincides with improved nucleosome positioning, heightened transcription factor binding, and increased expression of neighboring genes. Thus, ANP32E dramatically influences genome-wide chromatin accessibility through refinement of H2A.Z patterns, providing a means to reprogram chromatin state and to hone gene expression levels.

Download Full-text