A Stage-Specific Factor Confers Fab-7 Boundary Activity during Early Embryogenesis in Drosophila

ABSTRACT The Fab-7 boundary is required to ensure that the iab-6 and iab-7 cis-regulatory domains in the Drosophila Bithorax complex can function autonomously. Though Fab-7 functions as a boundary from early embryogenesis through to the adult stage, this constitutive boundary activity depends on subelements whose activity is developmentally restricted. In the studies reported here, we have identified a factor, called early boundary activity (Elba), that confers Fab-7 boundary activity during early embryogenesis. The Elba factor binds to a recognition sequence within a Fab-7 subelement that has enhancer-blocking activity during early embryogenesis, but not during mid-embryogenesis or in the adult. We found that the Elba factor is present in early embryos but largely disappears during mid-embryogenesis. We show that mutations in the Elba recognition sequence that eliminate Elba binding in nuclear extracts disrupt the early boundary activity of the Fab-7 subelement. Conversely, we find that early boundary activity can be reconstituted by multimerizing the Elba recognition site.

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The Fab-8 boundary defines the distal limit of the bithorax complex iab-7 domain and insulates iab-7 from initiation elements and a PRE in the adjacent iab-8 domain

Development ◽

10.1242/dev.127.4.779 ◽

2000 ◽

Vol 127 (4) ◽

pp. 779-790 ◽

Cited By ~ 12

Author(s):

S. Barges ◽

J. Mihaly ◽

M. Galloni ◽

K. Hagstrom ◽

M. Muller ◽

...

Keyword(s):

Higher Order ◽

Specific Pattern ◽

Bithorax Complex ◽

Response Element ◽

Regulatory Domains ◽

Polycomb Response Element ◽

Distal Side ◽

Blocking Activity ◽

Enhancer Blocking ◽

Order Domain

The Drosophila bithorax complex Abdominal-B (Abd-B) gene specifies parasegmental identity at the posterior end of the fly. The specific pattern of Abd-B expression in each parasegment (PS) determines its identity and, in PS10-13, Abd-B expression is controlled by four parasegment-specific cis-regulatory domains, iab-5 to iab-8, respectively. In order to properly determine parasegmental identity, these four cis-regulatory domains must function autonomously during both the initiation and maintenance phases of BX-C regulation. The studies reported here demonstrate that the (centromere) distal end of iab-7 domain is delimited by the Fab-8 boundary. Initiators that specify PS12 identity are located on the proximal iab-7 side of Fab-8, while initiators that specify PS13 identity are located on the distal side of Fab-8, in iab-8. We use transgene assays to demonstrate that Fab-8 has enhancer blocking activity and that it can insulate reporter constructs from the regulatory action of the iab-7 and iab-8 initiators. We also show that the Fab-8 boundary defines the realm of action of a nearby iab-8 Polycomb Response Element, preventing this element from ectopically silencing the adjacent domain. Finally, we demonstrate that the insulating activity of the Fab-8 boundary in BX-C is absolutely essential for the proper specification of parasegmental identity by the iab-7 and iab-8 cis-regulatory domains. Fab-8 together with the previously identified Fab-7 boundary delimit the first genetically defined higher order domain in a multicellular eukaryote.

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The Enhancer-Blocking Activity of the Fab-7 Boundary From the Drosophila Bithorax Complex Requires GAGA-Factor-Binding Sites

Genetics ◽

10.1534/genetics.104.029561 ◽

2004 ◽

Vol 168 (3) ◽

pp. 1371-1384 ◽

Cited By ~ 77

Author(s):

Susan Schweinsberg ◽

Kirsten Hagstrom ◽

Daryl Gohl ◽

Paul Schedl ◽

Ram P. Kumar ◽

...

Keyword(s):

Binding Sites ◽

Bithorax Complex ◽

Gaga Factor ◽

Factor Binding ◽

Blocking Activity ◽

Enhancer Blocking

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The bithorax complex iab-7 Polycomb Response Element has a novel role in the functioning of the Fab-7 chromatin boundary

10.1101/329284 ◽

2018 ◽

Cited By ~ 1

Author(s):

Olga Kyrchanova ◽

Amina Kurbidaeva ◽

Marat Sabirov ◽

Nikolay Postika ◽

Daniel Wolle ◽

...

Keyword(s):

Binding Sites ◽

Boundary Function ◽

Polycomb Group ◽

Homeotic Genes ◽

Bithorax Complex ◽

Response Element ◽

Regulatory Domains ◽

Polycomb Response Element ◽

Nuclear Extracts

AbstractExpression of the three Bithorax complex homeotic genes is orchestrated by nine parasegment-specific regulatory domains. Autonomy of each domain is conferred by boundary elements (insulators). Here, we have used an in situ replacement strategy to reanalyze the sequences required for the functioning of one of the best-characterized fly boundaries, Fab-7. It was initially identified by a deletion, Fab-71, that transformed parasegment (PS) 11 into a duplicate copy of PS12. Fab-71 deleted four nuclease hypersensitive sites, HS*, HS1, HS2, and HS3, located in between the iab-6 and iab-7 regulatory domains. Transgene and P-element excision experiments mapped the boundary to HS*+HS1+HS2, while HS3 was shown to be the iab-7 Polycomb response element (PRE). Recent replacement experiments showed that HS1 is both necessary and sufficient for boundary activity when HS3 is also presented in the replacement construct. Surprisingly, while HS1+HS3 combination has full boundary activity, we discovered that HS1 alone has only minimal function. Moreover, when combined with HS3, only the distal half of HS1, dHS1, is needed. A ∼1,000 kD multiprotein complex containing the GAF protein, called the LBC, binds to the dHS1 sequence and we show that mutations in dHS1 that disrupt LBC binding in nuclear extracts eliminate boundary activity and GAF binding in vivo. HS3 has binding sites for GAF and Pho proteins that are required for PRE silencing. In contrast, HS3 boundary activity only requires the GAF binding sites. LBC binding with HS3 in nuclear extracts, and GAF association in vivo depend upon the HS3 GAF sites, but not the Pho sites. Consistent with a role for the LBC in HS3 boundary activity, the boundary function of the dHS1+HS3mPho combination is lost when the flies are heterozygous for a mutation in the GAF gene. Taken together, these results reveal a novel function for the iab-7 PREs in chromosome architecture.Author SummaryPolycomb group proteins (PcG) are important epigenetic regulators of developmental genes in all higher eukaryotes. In Drosophila, these proteins are bound to specific regulatory DNA elements called Polycomb group Response Elements (PREs). PcG support proper patterns of homeotic gene expression throughout development. Drosophila PREs are made up of binding sites for a complex array of DNA binding proteins, including GAF and Pho. In the regulatory region of the bithorax complex (BX-C), the boundary/insulator elements organize the autonomous regulatory domains, and their active or repressed states are regulated by PREs. Here, we studied the domain organization of the Fab-7 boundary and the neighboring PRE, which separate the iab-6 and iab-7 domains involved in transcription of the Abd-B gene. It was previously thought that PRE recruits PcG proteins that inhibit activation of the iab-7 enhancers in the inappropriate domains. However, here we found that PRE contributes to boundary activity and in combination with a key 242 bp Fab-7 region (dHS1) can form a completely functional boundary. Late Boundary Complex (LBC) binds not only to dHS1 but also to PRE and is required for the boundary activity of both elements. At the same time, mutations of Pho binding sites strongly diminish recruiting of PcG but do not considerably affect boundary function, suggesting that these activities can be separated in PRE.

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Single cell RNA-seq reveals genes vital to in vitro fertilized embryos and parthenotes in pigs

Scientific Reports ◽

10.1038/s41598-021-93904-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Zhi-Qiang Du ◽

Hao Liang ◽

Xiao-Man Liu ◽

Yun-Hua Liu ◽

Chonglong Wang ◽

...

Keyword(s):

Embryo Development ◽

Gene Networks ◽

Regulatory Networks ◽

Rna Binding ◽

Expression Patterns ◽

Early Embryo ◽

Specific Factor ◽

Early Embryos ◽

Genome Activation

AbstractSuccessful early embryo development requires the correct reprogramming and configuration of gene networks by the timely and faithful execution of zygotic genome activation (ZGA). However, the regulatory principle of molecular elements and circuits fundamental to embryo development remains largely obscure. Here, we profiled the transcriptomes of single zygotes and blastomeres, obtained from in vitro fertilized (IVF) or parthenogenetically activated (PA) porcine early embryos (1- to 8-cell), focusing on the gene expression dynamics and regulatory networks associated with maternal-to-zygote transition (MZT) (mainly maternal RNA clearance and ZGA). We found that minor and major ZGAs occur at 1-cell and 4-cell stages for both IVF and PA embryos, respectively. Maternal RNAs gradually decay from 1- to 8-cell embryos. Top abundantly expressed genes (CDV3, PCNA, CDR1, YWHAE, DNMT1, IGF2BP3, ARMC1, BTG4, UHRF2 and gametocyte-specific factor 1-like) in both IVF and PA early embryos identified are of vital roles for embryo development. Differentially expressed genes within IVF groups are different from that within PA groups, indicating bi-parental and maternal-only embryos have specific sets of mRNAs distinctly decayed and activated. Pathways enriched from DEGs showed that RNA associated pathways (RNA binding, processing, transport and degradation) could be important. Moreover, mitochondrial RNAs are found to be actively transcribed, showing dynamic expression patterns, and for DNA/H3K4 methylation and transcription factors as well. Taken together, our findings provide an important resource to investigate further the epigenetic and genome regulation of MZT events in early embryos of pigs.

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A downstream-element-binding factor facilitates assembly of a functional preinitiation complex at the simian virus 40 major late promoter

Molecular and Cellular Biology ◽

10.1128/mcb.10.7.3635-3645.1990 ◽

1990 ◽

Vol 10 (7) ◽

pp. 3635-3645

Author(s):

D E Ayer ◽

W S Dynan

Keyword(s):

Rna Polymerase Ii ◽

Simian Virus 40 ◽

Initiation Site ◽

Simian Virus ◽

Recognition Sequence ◽

Base Pairs ◽

Transcriptional Initiation ◽

Nuclear Extracts ◽

Major Late Promoter ◽

Time Required

Recent work has shown that many promoters recognized by eucaryotic RNA polymerase II contain essential sequences located downstream of the transcriptional initiation site. We show here that the activity of a promoter element centered 28 base pairs downstream of the simian virus 40 major late initiation site appears to be mediated by a DNA-binding protein, which was isolated by affinity chromatography from HeLa cell nuclear extracts. In the absence of the other components of the transcriptional machinery, the protein bound specifically but weakly to its recognition sequence, with a Kd of approximately 10(-8) M. Analysis of kinetic data showed that mutation of the downstream element decreased the number of functional preinitiation complexes assembled at the promoter without significantly altering the time required for half the complexes to assemble. This suggests that in the absence of the downstream activating protein, preinitiation complexes are at least partially assembled but are not transcriptionally competent.

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Enhancer-Blocking Activity Is Associated with Hypersensitive Site V Sequences in the Human Growth Hormone Locus Control Region

DNA and Cell Biology ◽

10.1089/dna.2011.1268 ◽

2011 ◽

Vol 30 (12) ◽

pp. 995-1005 ◽

Cited By ~ 6

Author(s):

Yan Jin ◽

Kevin Oomah ◽

Peter A. Cattini

Keyword(s):

Growth Hormone ◽

Control Region ◽

Human Growth Hormone ◽

Human Growth ◽

Locus Control Region ◽

Hypersensitive Site ◽

Blocking Activity ◽

Enhancer Blocking

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Polypurine sequences within a downstream exon function as a splicing enhancer.

Molecular and Cellular Biology ◽

10.1128/mcb.14.2.1347 ◽

1994 ◽

Vol 14 (2) ◽

pp. 1347-1354 ◽

Cited By ~ 120

Author(s):

K Tanaka ◽

A Watakabe ◽

Y Shimura

Keyword(s):

Recognition Sequence ◽

Splice Site Selection ◽

Sequence Element ◽

Nuclear Extracts ◽

Gene Functions ◽

Mouse Immunoglobulin ◽

In Vitro Splicing ◽

Immunoglobulin Mu ◽

Splicing Enhancer

We have previously shown that a purine-rich sequence located within exon M2 of the mouse immunoglobulin mu gene functions as a splicing enhancer, as judged by its ability to stimulate splicing of a distant upstream intron. This sequence element has been designated ERS (exon recognition sequence). In this study, we investigated the stimulatory effects of various ERS-like sequences, using the in vitro splicing system with HeLa cell nuclear extracts. Here, we show that purine-rich sequences of several natural exons that have previously been shown to be required for splicing function as a splicing enhancer like the ERS of the immunoglobulin mu gene. Moreover, even synthetic polypurine sequences had stimulatory effects on the upstream splicing. Evaluation of the data obtained from the analyses of both natural and synthetic purine-rich sequences shows that (i) alternating purine sequences can stimulate splicing, while poly(A) or poly(G) sequences cannot, and (ii) the presence of U residues within the polypurine sequence greatly reduces the level of stimulation. Competition experiments strongly suggest that the stimulatory effects of various purine-rich sequences are mediated by the same trans-acting factor(s). We conclude from these results that the purine-rich sequences that we examined in this study also represent examples of ERS. Thus, ERS is considered a general splicing element that is present in various exons and plays an important role in splice site selection.

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Multiple interactions are involved in a highly specific association of the Mod(mdg4)-67.2 isoform with the Su(Hw) sites in Drosophila

Open Biology ◽

10.1098/rsob.170150 ◽

2017 ◽

Vol 7 (10) ◽

pp. 170150 ◽

Cited By ~ 4

Author(s):

Larisa Melnikova ◽

Margarita Kostyuchenko ◽

Varvara Molodina ◽

Alexander Parshikov ◽

Pavel Georgiev ◽

...

Keyword(s):

Protein Complexes ◽

Specific Interaction ◽

Btb Domain ◽

Rich Domain ◽

Blocking Activity ◽

Transgenic Lines ◽

Enhancer Blocking ◽

Specific Association ◽

Protein Variants ◽

Multiple Interactions

The best-studied Drosophila insulator complex consists of two BTB-containing proteins, the Mod(mdg4)-67.2 isoform and CP190, which are recruited to the chromatin through interactions with the DNA-binding Su(Hw) protein. It was shown previously that Mod(mdg4)-67.2 is critical for the enhancer-blocking activity of the Su(Hw) insulators and it differs from more than 30 other Mod(mdg4) isoforms by the C-terminal domain required for a specific interaction with Su(Hw) only. The mechanism of the highly specific association between Mod(mdg4)-67.2 and Su(Hw) is not well understood. Therefore, we have performed a detailed analysis of domains involved in the interaction of Mod(mdg4)-67.2 with Su(Hw) and CP190. We found that the N-terminal region of Su(Hw) interacts with the glutamine-rich domain common to all the Mod(mdg4) isoforms. The unique C-terminal part of Mod(mdg4)-67.2 contains the Su(Hw)-interacting domain and the FLYWCH domain that facilitates a specific association between Mod(mdg4)-67.2 and the CP190/Su(Hw) complex. Finally, interaction between the BTB domain of Mod(mdg4)-67.2 and the M domain of CP190 has been demonstrated. By using transgenic lines expressing different protein variants, we have shown that all the newly identified interactions are to a greater or lesser extent redundant, which increases the reliability in the formation of the protein complexes.

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