A Hox Transcription Factor Collective Binds a Highly Conserved Distal-less cis-Regulatory Module to Generate Robust Transcriptional Outcomes

Uhl, Juli D.; Zandvakili, Arya; Gebelein, Brian

doi:10.1371/journal.pgen.1005981

Cited by 46 publications

(75 citation statements)

References 71 publications

(97 reference statements)

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“…As described above, the Hox/HMP site only contributes to abdominal repression and not to thoracic activation [70]. This finding is consistent with previous studies that suggest that only posterior Hox factors mediate strong interactions with HMP proteins on Hox/HMP sites [26,44,45].…”

Section: Case Study: the Distal-less Conserved Regulatory Elementsupporting

confidence: 91%

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Mechanisms of Specificity for Hox Factor Activity

Zandvakili

Gebelein

2016

JDB

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Metazoans encode clusters of paralogous Hox genes that are critical for proper development of the body plan. However, there are a number of unresolved issues regarding how paralogous Hox factors achieve specificity to control distinct cell fates. First, how do Hox paralogs, which have very similar DNA binding preferences in vitro, drive different transcriptional programs in vivo? Second, the number of potential Hox binding sites within the genome is vast compared to the number of sites bound. Hence, what determines where in the genome Hox factors bind? Third, what determines whether a Hox factor will activate or repress a specific target gene? Here, we review the current evidence that is beginning to shed light onto these questions. In particular, we highlight how cooperative interactions with other transcription factors (especially PBC and HMP proteins) and the sequences of cis-regulatory modules provide a basis for the mechanisms of Hox specificity. We conclude by integrating a number of the concepts described throughout the review in a case study of a highly interrogated Drosophila cis-regulatory module named “The Distal-less Conserved Regulatory Element” (DCRE).

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Section: Case Study: the Distal-less Conserved Regulatory Elementsupporting

confidence: 91%

“…In the thorax, Antp cooperates with Exd/Hth to stimulate enhancer activity via binding the two Hox/PBC sites of the DCRE [70]. However, the Hox/HMP sites are not required for this activity, indicating that Antp may only specifically work on Hox/PBC sites and not Hox/HMP sites.…”

Section: Case Study: the Distal-less Conserved Regulatory Elementmentioning

confidence: 99%

Mechanisms of Specificity for Hox Factor Activity

Zandvakili

Gebelein

2016

JDB

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“…Cloning was facilitated by including flanking EcoR1 and BglII sequences. Annealed oligonucleotides or double stranded DNA fragments were cloned into either placZ-attB or 3xGBE-placZ-attB (Uhl, et al, 2016) and sequence confirmed. To concatenate 6xSPS into larger arrays, a shuttle vector was used to generate a BamH1-6xSPS-BglII-Not1 fragment for reiterative cloning into vectors digested with BglII/NotI (BglII/NotI permits cloning a BamH1/NotI fragment, which can be repeated as desired).…”

Section: Methodsmentioning

confidence: 99%

“…Purified proteins were confirmed by SDS-PAGE with Coomassie blue staining and concentrations were measured by absorbance at UV280 with calculated extinction coefficients. Electromobility shift assays (EMSAs) were performed as previously described using native polyacrylamide gel electrophoresis (Uhl, et al, 2016;Uhl, et al, 2010). Fluorescent labeled probes (3.4nM) were mixed with 22 increasing concentrations of purified CSL (RBPJ) protein in 2-fold steps (from 9.4 to 300nM) with or without purified NICD and Mastermind (Mam) proteins at 550nM.…”

Section: Protein Purification and Electrophoretic Mobility Shift Assamentioning

confidence: 99%

Enhancer architecture sensitizes cell specific responses toNotchgene dose via a bind and discard mechanism

Kuang

Golan

Preusse

et al. 2019

Preprint

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SUMMARYNotch pathway haploinsufficiency can cause severe developmental syndromes with highly variable penetrance. Currently, we have a limited mechanistic understanding of phenotype variability due to gene dosage. Here, we show that inserting a single enhancer containing pioneer transcription factor sites coupled to Notch dimer sites can unexpectedly induce a subset of Drosophila Notch haploinsufficiency phenotypes in an animal with wild type Notch gene dose. Mechanistically, this enhancer couples Notch DNA binding to degradation in a Cdk8-dependent, transcription-independent manner. Using mathematical modeling combined with quantitative trait and expression analysis, we show that tissues requiring long duration Notch signals are more sensitive to perturbations in Notch degradation compared to tissues relying upon short duration processes. These findings support a novel “bind and discard” mechanism in which enhancers with specific binding sites promote rapid Notch turnover, reduce Notch-dependent transcription at other loci, and thereby sensitize tissues to gene dose based upon signal duration.

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“…The activation of rho, which encodes a serine protease that triggers the release of an EGF ligand, results in the induction of neighboring cells to form an essential set of hepatocyte-like cells known as oenocytes [29][30][31][32]. In contrast, the Distal-less Conserved Regulatory Element (DCRE) contains three Hox/co-factor binding sites that recruit Abd-A/Exd/Hth complexes to repress Distal-less (Dll) gene expression in the abdominal ectoderm [33][34][35]. Dll, which is an appendage selector gene that promotes leg formation in thoracic segments, is thereby restricted from the abdomen to block appendage formation in these segments [36].…”

Section: Introductionmentioning

confidence: 99%

Thecis-regulatory logic underlying abdominal Hox-mediated repression versus activation of regulatory elements inDrosophila

Zandvakili

Uhl

Campbell

et al. 2018

Preprint

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Hox genes encode a family of transcription factors that, despite having similar in vitro DNA binding preferences, regulate distinct genetic programs along the metazoan anterior-posterior axis. To better define mechanisms of Hox specificity, we compared and contrasted the ability of abdominal Hox factors to regulate two cis-regulatory elements within the Drosophila embryo. Both the Ultrabithorax (Ubx) and Abdominal-A (Abd-A) Hox factors form cooperative complexes with the Extradenticle (Exd) and Homothorax (Hth) transcription factors to repress the distal-less leg selector gene via the DCRE, whereas only Abd-A interacts with Exd and Hth on the RhoA element to activate a rhomboid serine protease gene that stimulates Epidermal Growth Factor secretion.By swapping binding sites between these elements, we found that the RhoA Exd/Hth/Hox site configuration that mediates Abd-A specific activation can also convey transcriptional repression by both Ubx and Abd-A when placed into the DCRE, but only in one orientation. We further show that the orientation and spacing of Hox sites relative to additional transcription factor binding sites within the RhoA and DCRE elements is critical to mediate appropriate cell-and segment-specific output. These results indicate that the interaction between Hox, Exd, and Hth neither determines activation vs repression specificity nor defines Ubx vs Abd-A specificity. Instead the precise integration of Hox sites with additional TF inputs is required for accurate transcriptional output.Taken together, these studies provide new insight into the mechanisms of Hox target and regulatory specificity as well as the constraints placed on regulatory elements to convey appropriate outputs. Author SummaryThe Hox genes encode a family of transcription factors that give cells within each region along the developing body plan a unique identity in animals from worms to mammals. Surprisingly, however, most of the Hox factors bind the same or highly similar DNA sequences. These findings raise a paradox: How can proteins that have highly similar DNA binding properties perform different functions in the animal by regulating different sets of target genes? In this study, we address this question by studying how two Hox factors regulate the expression of target genes that specify leg development and the making of liver-like cells in the developing fly. By comparing and contrasting how Hox target genes are activated and/or repressed, we found that the same Hox binding sites can mediate either activation or repression in a manner that depends upon context. In addition, we found that a Hox binding site that is normally regulated by only one Hox factor, can also be used by more than one Hox factor swapped into another target gene. These findings indicate that the specificity of a Hox factor to regulate target genes does not rely solely upon DNA binding specificity but also requires regulatory specificity.

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A Hox Transcription Factor Collective Binds a Highly Conserved Distal-less cis-Regulatory Module to Generate Robust Transcriptional Outcomes

Cited by 46 publications

References 71 publications

Mechanisms of Specificity for Hox Factor Activity

Mechanisms of Specificity for Hox Factor Activity

Enhancer architecture sensitizes cell specific responses toNotchgene dose via a bind and discard mechanism

Thecis-regulatory logic underlying abdominal Hox-mediated repression versus activation of regulatory elements inDrosophila

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