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Single-stranded DNA binding proteins are essential components of the architectural LDB1 protein complex [4C-seq]

ABSTRACT: Transcriptional enhancers are brought into proximity with promoters via chromatin looping. The architectural transcription cofactor LDB1 facilitates spatial connectivity among enhancers and promoters, but whether this occurs through simple dimerization or requires heterotypic protein assemblies is unknown. Here we investigated single-stranded DNA binding proteins (SSBPs) in regulating LDB1-mediated chromatin looping and transcription. SSBP2, SSBP3, and SSBP4 colocalize with LDB1 genome-wide. Among these, only SSBP3 is essential for murine erythroid cell viability, LDB1 function, and transcription. LDB1 but not single-stranded DNA is the predominant genome-wide tether of SSBP3 to chromatin. SSBP3 depletion in SSBP2/4 knockout cells globally weakened LDB1-dependent chromatin loops and lowered nascent transcription without impacting LDB1's chromatin binding. Chromatin tethering experiments revealed SSBP3 and LDB1 mutually depend on each other to form looped contacts. SSBP3 stabilizes LDB1 dimerization in vitro, providing a possible mechanism. SSBPs emerge as key functional components of the architectural LDB1 complex.

ORGANISM(S): Mus musculus

PROVIDER: GSE288265 | GEO | 2025/07/23

REPOSITORIES: GEO

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Project description:Objective: Transcriptional complex activity drives the development and function of pancreatic islet cells to allow for proper glucose regulation. Our prior work highlighted that the LIM-homeodomain transcription factor (TF), Islet-1 (Isl1), and its interacting co-regulator, Ldb1, are vital effectors of developing and adult β-cells. We further found that a member of the Single Stranded DNA-Binding Protein (SSBP) co-regulator family, SSBP3, interacts with Isl1 and Ldb1 in β-cells and primary islets (mouse and human) to impact β-cell target genes MafA and Glp1R. Members of the SSBP family stabilize TF complexes by binding directly to Ldb1 and protecting the complex from ubiquitin-mediated turnover. In this study, we hypothesized that SSBP3 would have critical roles in pancreatic islet cell development and function in vivo, similar to the Isl1::Ldb1 complex. Methods: We first generated a novel SSBP3 LoxP allele mouse line, where Cre-mediated recombination imparts a predicted early protein termination. We bred this mouse to constitutive Cre lines (Pdx1- and Pax6-driven) to recombine of SSBP3 in the developing pancreas and islet (SSBP3DeltaPanc and SSBP3DeltaIslet), respectively. We assessed glucose tolerance and used immunofluorescence to detect changes in islet cell abundance and markers of β-cell identity and function. Using an inducible Cre system we also deleted SSBP3 in the adult β-cell, a model termed SSBP3Deltaβ-cell. We measured glucose tolerance as well as glucose-stimulated insulin secretion (GSIS), both in vivo and in isolated islets in vitro. Using islets from control and SSBP3Deltaβ-cell we conducted RNA-Seq and compared our results to published datasets for similar β-cell specific Ldb1 and Isl1 knockouts to identify commonly regulated target genes. Results: SSBP3DeltaPanc and SSBP3DeltaIslet neonates present with hyperglycemia. SSBP3DeltaIslet mice are glucose intolerant by P21 and exhibit a reduction of β-cell maturity markers MafA, Pdx1, and UCN3. We observe disruptions in islet cell architecture with an increase in glucagon+ α-cells and ghrelin+ ε-cells at P10. Inducible loss of β-cell SSBP3 in SSBP3Deltaβ-cell causes hyperglycemia, glucose intolerance, and reduced GSIS. Transcriptomic analysis of 14-week SSBP3Deltaβ-cell islets revealed a decrease in β-cell function gene expression (Ins, MafA, Ucn3), increased stress and dedifferentiation markers (Neurogenin-3, Aldh1a3, Gastrin), and shared differentially expressed genes between SSBP3, Ldb1, and Isl1 in adult β-cells. Conclusions: SSBP3 drives proper islet development and identity, where its loss causes altered islet-cell abundance and glucose regulatory function. β-cell SSBP3 is required for GSIS and glucose homeostasis, at least partially through shared regulation of Ldb1 and Isl1 target genes.

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Single-stranded DNA binding proteins are essential components of the architectural LDB1 protein complex [4C-seq]

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