SSBP3 Interacts With Islet-1 and Ldb1 to Impact Pancreatic β-Cell Target Genes

Abstract

Islet-1 (Isl1) is a Lin11, Isl1, Mec3 (LIM)-homeodomain transcription factor important for pancreatic islet cell development, maturation, and function, which largely requires interaction with the LIM domain-binding protein 1 (Ldb1) coregulator. In other tissues, Ldb1 and Isl1 interact with additional factors to mediate target gene transcription, yet few protein partners are known in β-cells. Therefore, we hypothesize that Ldb1 and Isl1 participate in larger regulatory complexes to impact β-cell gene expression. To test this, we used cross-linked immunoprecipitation and mass spectrometry to identify interacting proteins from mouse β-cells. Proteomic datasets revealed numerous interacting candidates, including a member of the single-stranded DNA-binding protein (SSBP) coregulator family, SSBP3. SSBPs potentiate LIM transcription factor complex activity and stability in other tissues. However, nothing was known of SSBP3 interaction, expression, or activity in β-cells. Our analyses confirmed that SSBP3 interacts with Ldb1 and Isl1 in β-cell lines and in mouse and human islets and demonstrated SSBP3 coexpression with Ldb1 and Isl1 pancreas tissue. Furthermore, β-cell line SSBP3 knockdown imparted mRNA deficiencies similar to those observed upon Ldb1 reduction in vitro or in vivo. This appears to be (at least) due to SSBP3 occupancy of known Ldb1-Isl1 target promoters, including MafA and Glp1r. This study collectively demonstrates that SSBP3 is a critical component of Ldb1-Isl1 regulatory complexes, required for expression of critical β-cell target genes.

Figure 1.

Ldb1 and Isl1 enrich for SSBP3 in mouse βTC-3 cells. A, βTC-3 nuclear extracts were separated by 10%–35% sucrose gradient centrifugation and Western blotted for Ldb1, Isl1, p300, and Pdx1. A large portion of the Ldb1 and Isl1 protein signal was found in higher density fractions (eg, number 18) representing large protein complexes (ie, p300 containing). B, Workflow for the ReCLIP experiments. Briefly, βTC-3 cells were incubated with the reversible DSP cross-linker, then nuclear extracts were prepared and incubated with Ldb1- or Isl1-specific antibodies, or species-matched IgG controls. IP complexes were washed with RIPA buffer and eluted with RIPA supplemented with DTT to reduce the cross-linking. C, As confirmation, ReCLIP eluates were tested for known interaction between Isl1 and Ldb1 (left) and then visualized by SDS-PAGE followed by silver staining (right), before mass spectrometry analyses. D, Venn diagram depicting relative numbers of enriched peptides in Isl1 (red) or Ldb1 (green) mass spectrometry datasets after comparison with IgG. SSBP3 was a candidate factor enriched by both Ldb1 and Isl1. E, Confirmatory IP results from βTC-3 nuclear extract without cross-linking. Ldb1 or Isl1 IP recovers SSBP3 (top), as compared with input positive control and IgG IP negative control. Reciprocal IP of SSBP3 also enriched for Ldb1 and Isl1 (bottom). F, SSBP3 IP does not enrich for Pdx1, Pax6, or MafA TF proteins in βTC-3 cells, suggesting specificity with Ldb1 and Isl1.

Figure 2.

SSBP3 is coexpressed with Isl1 and Ldb1 in developing and adult islets. A–C, Immunofluorescence analyses with E13.5 (A), E18.5 (B), and 9-month-old (C) pancreas tissue indicate that SSBP3 (blue), Ldb1 (red), and Isl1 (green) are coexpressed during development and in adult islets. D–F, SSBP3 (blue) is also expressed in E15.5 Ngn3⁺/Ldb1⁺ endocrine progenitors (D) and in developing (E) and adult islet (F) insulin⁺ and glucagon⁺ cells. Yellow arrowheads point to Isl1⁺/Ldb1⁺/SSBP3⁺ nuclei in A, and Ngn3⁺/Ldb1⁺/SSBP3⁺ nuclei in D. Dashed yellow lines encircle SSBP3⁺ cell clusters coexpressing Ldb1 and Isl1 (B) or insulin and glucagon (E). G, Western blot analysis of mouse β-cell line (βTC-3, Min6) and nonpancreatic cell line (NIH-3T3) nuclear extracts. SSBP3, Ldb1, and Isl1 were enriched in β-cell extracts as compared with NIH-3T3. The related SSBP2 protein appears to be expressed at lower levels in only the βTC-3 extract. Actin is included as loading control (bottom). The arrowheads point to the immuno-specific band in the SSBP3 and SSBP2 blots. H, qPCR was performed to measure relative SSBP2, SSBP3, and SSBP4 mRNA levels in Min6 (gray bars) and βTC-3 (black bars) cells. Expression levels are displayed relative to TATA-binding protein (TBP), set as 1-fold. Error bars represent ±SEM (n = 3 for each cell line). ##, P < .01 comparing SSBP2 and SSBP3 mRNA levels; **, P < .01 comparing SSBP4 and SSBP3 mRNA levels.

Figure 3.

SSBP3 is required for Ldb1 target gene activation. A, siRNA-mediated knockdown reduces SSBP3 (left) or Ldb1 (right) protein levels, respectively. Two independent knockdown transfections into Min6 β-cells demonstrate SSBP3 or Ldb1 protein reduction, as compared with actin loading control. B and C, Comparative siSSBP3 (black bars) or siLdb1 (gray bars) knockdown in Min6 cells followed by RNA extraction, cDNA synthesis and qPCR analysis. Notably, several TF (B) or GSIS gene mRNAs (C) were significantly reduced by SSBP3 or Ldb1 knockdown; *, P < .05. mRNA levels were normalized to the gapdh housekeeping gene and compared with siSCR control (horizontal black line at 1-fold). Error bars represent ±SEM (n = 4–6 for each knockdown). D, Luciferase assay from siRNA and reporter plasmid cotransfected Min6 cell extracts. Empty pFox-LUC vector or MafA Region 3-LUC plasmids bearing a WT (TAAT) or mutant (MUT) (GCCG) Isl1-binding element were cotransfected with either siSCR or siSSBP3. WT MafA Region 3-LUC cotransfected with siSCR was set to 1-fold activation. SSBP3 knockdown significantly reduced WT MafA Region 3 activity; ****, P < .0001; ns, not significant. E, Western blot analysis of LUC plasmid and siRNA cotransfected Min6 extracts used in panel D confirm SSBP3 protein reduction while also demonstrating reduced Ldb1 and Isl1 protein levels in SSBP3 knockdown extracts. Actin was the loading control. The arrow points to the SSBP3-specific immunoreactive bands.

Figure 4.

SSBP3 occupies 5′ domains of known Ldb1 target genes. A and B, ChIP analysis of SSBP3 binding to the MafA Region 3 control domain (A) as well as a Glp1r 5′ domain (B). Results are presented as amplified PCR products separated by standard agarose gel electrophoresis (left) or qPCR (right). The inactive albumin promoter served as negative background control, whereas H₂O was the PCR negative control. qPCR results were compared with species-matched IgG control ChIP and set to 1-fold. ChIP enrichment P values for both target genes are listed and less than 0.05 (*).

Figure 5.

SSBP3 interacts with Ldb1 and Isl1 in mouse and human islets. A, co-IP was performed with protein extracts isolated from WT mouse islets. Ldb1 IP enriched for both SSBP3 (top) and Isl1 (bottom) in the IP eluates, as compared with the IgG control IP. Conversely, SSBP3 (arrowhead) and Isl1 immunoreactive bands are depleted from the IP supernatant (S/N) but remain in the IgG control supernatant. B, SSBP3 was reduced by siRNA transfection into isolated then gently dispersed mouse islets, as compared with scrambled siRNA control (set to 1-fold enrichment, solid black line). MafA and Hb9 target mRNAs were significantly reduced (*, P < .05), whereas Glp1r levels trended (P = .12) toward reduction upon SSBP3 knockdown (**, P < .01). C, co-IP was performed with protein extracts isolated from human donor islets, as in panel A. LDB1 IP enriched for both SSBP3 and ISL1. D, Immunofluorescence analysis of human donor pancreas tissue indicates that LDB1 (red), ISL1 (green), and SSBP3 (blue) are coexpressed and highly enriched in human islet cells. Yellow arrowheads point to ISL1⁺ cells coexpressing LDB1 and SSBP3.