Journal: Genome biology

Article Title: BORIS/CTCFL epigenetically reprograms clustered CTCF binding sites into alternative transcriptional start sites.

doi: 10.1186/s13059-024-03175-0

Figure Lengend Snippet: Fig. 7 Opening of chromatin by BORIS facilitates binding of other transcriptional factors. a Scatter plot displaying the enrichment of 190 TF motifs at CTCF/BORIS binding sites that reprogrammed into active promoters, compared to transcriptionally silent CTCF/BORIS sites in NIH3T3 + BORIS (clone#2) cells. b MAZ and MXI1 motifs are significantly enriched at CTCF/BORIS binding sites converted into active promoters. c Genome browser view illustrates the recruitment of TBP, HCFC1, MXI1, and MAZ proteins at the CTCF site within the Rbpjl promoter, activated by BORIS binding in NIH3T3 + BORIS (clone#2) cells. The activated promoter is highlighted by a red open box. d Left panel: Scatter plot of normalized read counts (log10) for HCFC1 occupancy at the combined set of HCFC1 binding sites (46,287) in NIH3T3 + BORIS (clone#2) cells compared to the same genomic sites in EV cells. Right panel: Heatmap of CTCF (red), BORIS (blue), and HCFC1 (brown) occupancy at the 19,519 HCFC1 sites from the left panel (connected by red arrow). e TF motifs enriched at HCFC1 peaks (60 bp around the summit of peak) in NIH3T3 + EV versus NIH3T3 + BORIS (clone#2) cells. f Heatmap of BORIS (blue), TBP (purple), HCFC1 (brown), MXI1 (orange), and MAZ (green) occupancy at the 5871 CTCF/BORIS binding sites converted into active promoters in NIH3T3 + BORIS (clone#2) cells compared to NIH3T3 + EV cells from Fig. 4h. g Summary of epigenetic reprogramming: BORIS binding recruits SRCAP, which replaces H2A histone with H2A.Z, leading to the opening of chromatin around CTCF sites. This, in turn, attracts other TFs to bind and stimulate transcription, resulting in the conversion of transcriptionally inert CTCF sites into active promoters

Article Snippet: Rabbit polyclonal antibody against SRCAP (Kerafast, ESL103), rabbit polyclonal against HCFC1 (Novus Biologicals, NB100-68209), goat affinity-purified polyclonal antibody against Mxi1 (R&D Systems, AF4185), rabbit monoclonal to TATA-binding protein TBP (Abcam, ab220788), rabbit polyclonal anti-PHF8 antibody (Bethyl Laboratories, A301-772A), rabbit polyclonal to MAZ (Abcam, ab85725), rabbit polyclonal antibody against the region of histone H3 containing the trimethylated lysine 27 (H3K27me3) (Diagenode, C15410195), rabbit polyclonal antibody against histone H3, trimethylated at lysine 36 (H3K36me3) (Diagenode, C15410058), rabbit polyclonal antibody against histone H2A.Z (Abcam, ab4174), rabbit polyclonal to Histone H3 (acetyl K27) (Abcam, ab4729), rabbit polyclonal to Histone H3 (tri methyl K4) (Abcam, ab8580), anti-RNA Polymerase II Antibody, CTD Antibody, clone 8WG16 (Millipore, 05–952-I-100UG).

Techniques: Binding Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Directing differentiation of human induced pluripotent stem cells toward androgen-producing Leydig cells rather than adrenal cells.

doi: 10.1073/pnas.1908207116

Figure Lengend Snippet: Fig. 1. Induction of human Leydig-like cells (hLLCs) and human adrenal-like cells (hALCs) from human induced pluripotent stem cells (hiPSCs). (A) Schematic of differentiation stages from hiPSCs into human adrenal cells (hACs) or human Leydig cells (hLCs). Genes displayed in each stage represent cellular markers of that stage. EMP, early mesenchymal progenitors. (B) Schematic strategy for the induction hALCs and hLLCs from hiPSCs. Before the specification of hALCs and hLLCs, hiPSCs were first induced into EMPs. At 2 d before the EMP induction, hiPSCs were plated on Matrigel-coated plates and cultured in mTeSR medium for pro- liferation. On induction day (ID) 0, cells were cultured in STEMdiff-ACF Mesenchymal Induction (ACF-MI) medium for 4 d and MesenCult-ACF (MC-ACF) medium for 2 d to form EMPs. (Top) hALCs were induced from EMPs by the ACF-SF1 system. On ID 6, EMPs were transferred to MesenCult-ACF Attachment Substrate-coated plates and cultured in MC-ACF medium. On ID 12, EMPs were transfected with SF-1 plasmid and exposed to dibutyryl-cAMP (dbcAMP) and desert hedgehog (DHH) in MC-ACF medium for 6 d. On ID 18, cells were exposed to dbcAMP, DHH, and human CG (hCG) in MC-ACF medium for 8 d. On ID 26, hALCs formed. (Bottom) hLLCs were induced from EMPs by the COLI-SF1 system. On ID 6, EMPs were transferred to Collagen type I (COLI)-coated plates and cultured in F12/DMEM supplemented with 10% FBS (F12/FBS) for 2 d. On ID 8, cells were transfected with SF-1 plasmid and exposed to dbcAMP and DHH in F12/FBS medium for 6 d. On ID 14, cells were exposed to dbcAMP, DHH, and hCG in F12/FBS medium for 8 d. On ID 22, hLLCs formed. (C) qRT-PCR analyses of hiPSC markers (OCT4 and NANOG), primitive streak markers (MIXL1 and TBXT), and EMP markers (PDGFRA and COUP-TFII) from hiPSCs and EMPs. The low expression of OCT4, NANOG, MIXL1, and TBXT indicate that EMPs have lost pluripotency and passed the primitive streak stage. The high expression of PDGFRA and COUP-TFII indicates the appearance of EMPs. (D and F) qRT-PCR analyses of steroidogenic cell markers in hiPSCs, hALCs, and hLLCs, respectively. Both hALCs and hLLCs highly expressed SF-1. hALCs highly expressed many steroidogenic genes but not the adrenal-specific gene CYP21B and testis-specific genes HSD17B3 and CYP19A1, in comparison to hiPSCs. The insignificant up-regulation of CYP21B in hALCs might be due to its detectable expression level in both hiPSCs and hALCs that is shown in SI Appendix, Fig. S2A, suggesting gene expression in hALCs is similar to that of hACs. hLLCs highly expressed most of the steroidogenic genes except for CYP21B, suggesting their gene expression is similar to that of hLCs. (E and G) ELISAs measuring cortisol (CORT), aldosterone (ALDO), and testosterone (T) in cell supernatants of hALCs and hLLCs, respectively. hALCs produced significantly more CORT and ALDO than T, while hLLCs produced significantly more T than CORT and ALDO. NC, GFP-transfected negative control cells. The quantitative comparison of T and CORT produced by hALCs and hLLCs is shown in SI Appendix, Fig. S2 D and E. Data in C–G are presented as mean ± SD, n = 3. In D and F, P value was generated by ANOVA. Multiple comparisons were corrected by Tukey’s t test. In E and G, P value was generated by the Student’s t test. n.s., not significant at P > 0.05 (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).

Article Snippet: A total of 1 μg of Steroidogenic Factor 1 (NR5A1) human-tagged ORF cloning vector (Origene, RC207577L2) was transfected into the cells using Lipofectamine 3000 and Opti-MEM according to the manufacturer’s instruction. pLenti-C-mGFP–tagged cloning vector (Origene, PS100071) was used as the GFP-transfected negative control.

Techniques: Cell Culture, Transfection, Plasmid Preparation, Quantitative RT-PCR, Expressing, Comparison, Gene Expression, Produced, Negative Control, Generated

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Directing differentiation of human induced pluripotent stem cells toward androgen-producing Leydig cells rather than adrenal cells.

doi: 10.1073/pnas.1908207116

Figure Lengend Snippet: Fig. 3. Protein expression profile and ultrastructure of hLLCs. (A) Immunocytochemistry analyses of hLC marker HSD3B2 and adult LC marker HSD17B3. Cells with positive stains were indicated by arrows. The table next to them showed that the percentages of HSD3B2- and HSD17B3-positive cells were 93.08 ± 4.21% and 57.02 ± 9.47%, respectively. (Scale bar, 100 μm.) (B) Immunofluorescent staining of HSD3B2 (green channel) indicated that the vast majority of cells differentiated toward hLLCs. Some hLLCs also expressed LHCGR (red channel, indicated by arrow), suggesting that the function of some hLLCs is regulated by hCG/LH. (Scale bar, 100 μm.) (C) Western blot analyses of SF-1, CYP11A1, HSD3B2, CYP17A1, LHCGR, STAR, and GAPDH in hiPSCs and hLLCs. The results in- dicate that, upon the overexpression of SF-1, hLLCs highly expressed CYP11A1, HSD3B2, and CYP17A1, all of which are important for T biosynthesis. The presence of LHCGR and STAR implies that hLLCs could transfer cholesterol to mitochondria under the regulation of hCG/LH signaling. Human testicular lysate was used as a positive control. (D) Western blot analyses of HSD17B3, CYP21B, and OCT4 in hiPSCs, hLLCs, and hALCs. The specific expression of HSD17B3 in hLLCs indicates the hLC properties of hLLCs. The negligible expression of CYP21B in hLLCs suggests their poor synthetic capacity for CORT and ALDO. The undetectable expression of OCT4 in hLLCs and hALCs suggests that hiPSCs stemness had been eliminated. (E–H) Transmission electron microscopy images of hLLCs showing their ultrastructure. (E–F) hLLCs possessed condensed smooth endoplasmic reticulum (SER; indicated by arrow). (G) The swirled variety of SER (WER) is also found in hLLCs. (G and H) The presence of plentiful mitochondria (M) that were close together. Note that their cristae were more lamellar-like. Moderate amounts of lipid droplets (LDs), rough endoplasmic reticulum (RER), and Golgi (G) were also found in hLLCs. N, nucleus. (Scale bars: E and G, 500 nm; F and H, 200 nm.) The full-size TEM images are provided in SI Appendix, Figs. S9–S12.

Article Snippet: A total of 1 μg of Steroidogenic Factor 1 (NR5A1) human-tagged ORF cloning vector (Origene, RC207577L2) was transfected into the cells using Lipofectamine 3000 and Opti-MEM according to the manufacturer’s instruction. pLenti-C-mGFP–tagged cloning vector (Origene, PS100071) was used as the GFP-transfected negative control.

Techniques: Expressing, Immunocytochemistry, Marker, Staining, Western Blot, Over Expression, Positive Control, Transmission Assay, Electron Microscopy

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Directing differentiation of human induced pluripotent stem cells toward androgen-producing Leydig cells rather than adrenal cells.

doi: 10.1073/pnas.1908207116

Figure Lengend Snippet: Fig. 4. Steroid production in hLLCs and hALCs. (A) Steroidogenic pathway in hALCs and hLLCs leading to the production of ALDO, CORT, and T. P5, preg- nenolone; 17α-hydroxy-P5, 17α-hydroxy-pregnenolone; DHEA, dehydroepian- drosterone; P4, progesterone; 17α-Hydroxy-P4, 17α-hydroxy-progesterone; A4, androstenedione; 11-Deoxy-B, 11-deoxycorticosterone; B, corticosterone; 11-deoxy-CORT, 11-deoxycortisol. (B–E) ELISA analyses of P5, P4, DHEA, and A4 in the media of SF-1–overexpressing hALCs and hLLCs. Compared with hALCs, hLLCs produce similar amounts of P5 (B) and less P4 (C), but greater amounts of DHEA and A4 (D and E). NC, negative controls. Data are presented as mean ± SD, n ≥3. P value was generated by the Student’s t test. n.s., not significant at P > 0.05 (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).

Article Snippet: A total of 1 μg of Steroidogenic Factor 1 (NR5A1) human-tagged ORF cloning vector (Origene, RC207577L2) was transfected into the cells using Lipofectamine 3000 and Opti-MEM according to the manufacturer’s instruction. pLenti-C-mGFP–tagged cloning vector (Origene, PS100071) was used as the GFP-transfected negative control.

Techniques: Enzyme-linked Immunosorbent Assay, Generated