Biology subjects

Haro, E.

Publications and source records attributed to Haro, E..

4 recordsLinked to original sources

bioRxiv2025

Synthetic engineering demonstrates that synergy among enhancers involves an increase in transcriptionally productive enhancer-gene contacts

Enhancers are non-coding cis-regulatory elements that control the expression of distally located genes in a tissue- and time-specific manner. Recent studies indicate that enhancers can differ in their underlying genetic architecture and regulatory properties. However, these different types of enhancers were previously investigated under rather variable conditions (e.g. model organism, cell type, enhancer-promoter distance, type of target promoter, etc.), thus introducing confounding factors that make it difficult to discern the distinct regulatory properties of each enhancer type. To overcome these limitations, here we generated transgenic mouse embryonic stem cells (mESC) lines in which different types of synthetic enhancers (i.e. "typical" enhancer, CTCF-associated enhancer, enhancer cluster/super-enhancer) were built upon the same "core" neural enhancer and inserted at the same distance (i.e. 100 Kb) from a typical developmental gene (i.e. Gata6). Subsequently, the mESC lines were differentiated to systematically compare the regulatory properties of the different enhancer types under identical conditions. Regarding the CTCF-associated enhancer, our data revealed that the addition of a CTCF site to the "core" enhancer increased insulation and led to the formation of a smaller contact domain, while having a rather mild effect on enhancer-promoter contact frequency and target gene expression. On the other hand, in comparison to the "core" enhancer alone, the enhancer cluster synergistically increased target gene expression and burst fraction. Importantly, we found that, in contrast to previous models, the strong regulatory activity of the enhancer cluster can not be explained by changes in enhancer-promoter contact frequency or the formation of transcriptional condensates. Instead, our data suggest that the emergent regulatory properties of enhancer clustering preferentially entail an increase in RNA Polymerase II pause release and, thus, in the fraction of enhancer-promoter contacts that are transcriptionally productive.

genetics↗

bioRxiv2023

Synergistic insulation of regulatory domains by developmental genes and clusters of CTCF sites

The specificity of gene expression during development requires the insulation of regulatory domains to avoid inappropriate enhancer-gene interactions. In vertebrates, this insulator function is mostly attributed to clusters of CTCF sites located at topologically associating domain (TAD) boundaries. However, TAD boundaries allow a certain level of physical crosstalk across regulatory domains, which is at odds with the highly specific and precise expression of developmental genes. Here we show that developmental genes and nearby clusters of CTCF sites synergistically foster the robust insulation of regulatory domains. Firstly, we found that the TADs containing developmental genes have distinctive features, including the sequential organization of developmental genes and CTCF clusters near TAD boundaries. Most importantly, by genetically dissecting representative loci in mouse embryonic stem cells, we showed that developmental genes and CTCF sites synergistically strengthened the insulation capacity of nearby boundaries through different mechanisms. Namely, while CTCF sites prevent undesirable enhancer-gene contacts (i.e. physical insulation), developmental genes preferentially contribute to regulatory insulation through non-structural mechanisms involving promoter competition rather than enhancer blocking. Overall, our work provides important insights into the specificity of gene regulation, which in turn might help interpreting the pathological consequences of certain structural variants.

genetics↗

bioRxiv2022

Failure of digit tip regeneration in the absence of suggests Lmx1b functions disparate from dorsoventral polarity

Mammalian digit tip regeneration is linked to the presence of nail tissue, but a nail-explicit model is missing. Here, we report that nail-less double-ventral digits of {Delta}LARM1/2 mutants that lack limb-specific Lmx1b enhancers fail to regenerate. To separate the nails effect from the lack of DV polarity, we also interrogate double-dorsal double-nail digits and show that they regenerate. Thus, DV polarity is not a prerequisite for regeneration and the nail requirement is supported. Transcriptomic comparison between wild-type and non-regenerative{Delta} LARM1/2 mutant blastemas reveals differential up-regulation of vascularization and connective tissue functional signatures in wild-type versus upregulation of inflammation in the mutant. These results, together with the finding of uniform Lmx1b expression in the wild-type blastema and in the dorsal dermis underneath the nail, indicate that, in addition of the nails effect, a direct role for Lmx1b in driving the progression of digit tip regeneration is likely.

developmental biology↗

bioRxiv2020

Identification of limb-specific Lmx1b auto-regulatory modules with Nail-Patella Syndrome pathogenicity.

LMX1B haploinsufficiency causes Nail-patella syndrome (NPS; MIM 161200), characterized by nail dysplasia, absent/hypoplastic patellae, chronic kidney disease, and glaucoma. Accordingly, in mice Lmx1b has been shown to play crucial roles in the development of the limb, kidney and eye. Although one functional allele of murine Lmx1b appears adequate for development, Lmx1b null mice display ventral-ventral distal limbs with abnormal kidney, eye and cerebellar development, more disruptive, but fully concordant with NPS. Interestingly, in Lmx1b functional knockouts (KOs), Lmx1b transcription in the limb is decreased nearly 6-fold indicating autoregulation. Herein, we report on two conserved Lmx1b-associated cis-regulatory modules (LARM1 and LARM2) that are bound by Lmx1b, amplify Lmx1b expression in the limb and are necessary for Lmx1b-mediated limb dorsalization. Remarkably, we also report on two NPS patient families with normal LMX1B coding sequence, but loss-of-function variations in the LARM1/2 region, stressing the role of regulatory modules in disease pathogenesis.

developmental biology↗