Dynamic regulation of pancreatic β cell function and gene expression by the SND1 coregulator in vitro

Figures & data

Table 1. gRNA sequence information.

lentiGuide-Puro sgRNA
gRNA Control Snd1 Intron	TTTTGGCTCAACCAACGCAAAGG
gRNA1 Snd1 Exon 2	GGTGCGCCATAATTGTCCGAGGG
gRNA 2 Snd1 Exon 2	AATTGTCCGAGGGCAGCCCCGGG

Table 2. nPOD donor sample information.

Case #:	RRID	Diabetes Status	Age (years)	Sex	Race	BMI (kg/m^2)	T2D Duration (years)	Clinical Information
6020	SAMN15879077	No diabetes	60	Male	Caucasian	29.8	-	intracerebral hemorrhage
6095	SAMN15879152	No diabetes	40	Male	Hispanic/Latino	35.5	-	traffic accident
6254	SAMN15879310	No diabetes	38	Male	Caucasian	30.5	-	anoxic brain injury
6168	SAMN15879224	No diabetes	51	Male	Hispanic/Latino	25.2	-	acute ischemic stroke
6297	SAMN15879351	T2D	60	Male	Caucasian	29.5	3	pulmonary embolism
6203	SAMN15879259	T2D	68.6	Male	Caucasian	32.5	5	intracerebral/intraventricular hemorrhage
6255	SAMN15879311	T2D	55	Male	Caucasian	29.35	6	intraparenchymal hemorrhage
6124	SAMN15879181	T2D	62.3	Male	Caucasian	33.7	3	stroke

Table 3. qPCR primers.

Gene	Forward Primer	Reverse Primer
Mouse qPCR primers
18s	AGTCCCTGCCCTTTGTACACA	GATCCGAGGGCCTCACTAAAC
Glp1r	ACGGTGTCCCTCTCAGAGAC	ATCAAAGGTCCGGTTGCAGAA
Pdgfra	AGAGTTACACGTTTGAGCTGTC	GTCCCTCCACGGTACTCCT
Rgs8	GCAGGAACAAAGGCATGAGGA	TGCTTCTTCCGTGGAGAGTCT
Rgs16	CCATGCCTTCCTAAAGACGGA	GTACTCGTCAAAGATGTGGTGAG
Sct	AGACACTCAGACGGAATGTTCA	CTGGTCCTCTAAGGGCTTGGA
Six2	CACCTCCACAAGAATGAAAGCG	CTCCGCCTCGATGTAGTGC
Pou3f2	GCAGCGTCTAACCACTACAGC	GCGGTGATCCACTGGTGAG
Sp8	GCTACCTGTAATAAGATCGGCAG	GAGGAGCGTTTCCAAGGGTG
Crp	ATGGAGAAGCTACTCTGGTGC	ACACACAGTAAAGGTGTTCAGTG
Human qPCR primers
18S	GCAGAATCCACGCCAGTACAAG	GCTTGTTGTCCAGACCATTGGC
PDX1	AAGTCTACCAAAGCTCACGCG	GTAGGCGCCGCCTGC
SND1	CAGAACCGGCTTTCAGAATGT	TAGTATGTGAACCGTTCCCCT
INS	AGAGGCCATCAAGCAGATCACTGT	AGGTGTTGGTTCACAAAGGCTG
GLP1R	GGTGCAGAAATGGCGAGAATA	CCGGTTGCAGAACAAGTCTGT
MAFA	TGAGCGGAGAACGGTGATTTCTAAGG	GGAACGGAGAACCACGTTCAACGTA
RGS16	ATCAGAGCTGGGCTGCGATA	CAGGTCGAACGACTCTCTCC
SIX2	CCTGCGAGCACCTTCACAA	CTCGATGTAGTGTGCCTTGAG
SLC2A1	GGACAGGCTCAAAGAGGTTATG	AGGAGGTGGGTGGAGTTAAT
SLC2A2	CTAGGCAGAGCTGCGAATAAA	CTAGTTGGGAGTCCTGTCAATTC
MAFB	ACCTTGGCTAAGGCGAGAGTAG	CTTCAGCCTGGAGAGAAGTTACTC

Figure 1. PDX1 and SND1 interact in rodent and human β cell lines and primary β cells. (a) co-immunoprecipitation of PDX1 or SND1, followed by immunoblotting for PDX1 and SND1 in INS-1 832/13 and βTC3 nuclear extracts. (b-d) proximity ligation assays (PLAs) using antibodies for SND1 and PDX1 were performed on (b) 12-week-old C57BL/6J, (c) human EndoC-βH1 β cells, and (d) 51-year-old non-diabetic male donor tissues acquired from nPOD (donor # 6168). The images on the right in b-d are magnified regions outlined in the yellow box. The white, fluorescent foci from the PLA represent individual PDX1:SND1 interactions. (scale bars = 10 μm).

Figure 2. Reduction in Snd1 negatively impacts glucose-stimulated insulin secretion in rodent and human β cell lines. Immunoblot of nuclear extracts from siControl and siSnd1 treated INS-1 832/13 (a) and EndoC-βH1 (d) cells demonstrate significant reductions of SND1. Quantitation of SND1 protein levels normalized to β-ACTIN shown to the right. (b) glucose-stimulated insulin secretion in siSnd1 cells is blunted in INS-1 832/13 (b) and EndoC-βH1 (e) cells with no change in insulin content (c, f). (n = 3–5). ns, not significant; *, p < 0.05; ****, p < 0.0001.

Figure 3. SND1 controls a subset of genes important for β cell expansion and function. (a) immunoblot of nuclear extracts for SND1 and β-ACTIN from clonal CRISPR/Cas9 intron - control and exon 2 - Snd1 KO cell lines. (b) volcano plot illustrating the most differentially expressed genes in Snd1 KO β cell lines. (c Heatmap hierarchical clustering displaying log2(Fold change) of select subset of differentially expressed genes in Snd1 KO β cells. (d) qPCR performed on independently isolated RNA from control and Snd1 KO cell lines. (e) biological processes of the 159 differentially expressed genes identified by gene ontology (GO) analysis include those associated with insulin secretion, cAMP biosynthesis, and cell proliferation. (f) qPCR of select subset of genes following siRNA-mediated depletion of SND1 from EndoC-βH1 cells. *, p < 0.05; **, p < 0.01.

Figure 4. PDX1:SND1 interactions are negatively impacted in T2D human β cells. (a) Representative PDX1:SND1 PLA and proinsulin images acquired from human pancreatic tissue sections from non-diabetic donor (nPOD case #: 6020: 60 years old male, BMI = 29.8) and T2D donor (nPOD case#: 6297: 60 years old male, BMI = 29.5, 3 years T2D duration). (b) quantitation of PLA signals in each group stratified by number of PLA signals per β cell nuclei. The images to the right in (a) are magnified regions outlined in the yellow box above. (n = 4). ns, not significant; *, p < 0.05; **, p < 0.01. (scale bars = 10 μm).