Fig 2: The transcription factor Creb5 is differentially expressed in superficial versus deep zone articular chondrocytes and is specifically expressed in Prg4+ cells in synovial joints.a RT-qPCR analysis of Prg4 and Creb5 expression in either SZCs or DZCs cultured in either the absence (white) or presence (gray) of TGF-ß2 (20 ng/ml) for 3 days. Gene expression was assayed by RT-qPCR and normalized to Gapdh. Error bar indicates standard error of the mean (n = 2 technical repeats). Similar results have been obtained in three independent biological repeats. b Western analysis of proteins in SZCs and DZCs. Similar results have been obtained in two independent biological repeats. c Immunofluorescent staining for CREB5, lubricin, and DAPI (to visualize nuclei) in adult human femoral head articular cartilage. Scale bar equals 50 microns. d Schematic of Prg4 expression in a developing long bone cartilage element. e Expression of Collagen 2a1 (Col2), Prg4, Creb5, and Matrilin1 (Matn1) in the elbows of P0 mice, as detected by fluorescent in situ hybridization. f Immunofluorescent staining for Creb5 protein and DAPI (to visualize nuclei) in the knee joint of a P0 mouse. In (e, f), the Creb5-expressing articular perichondrium is designated by a yellow arrow, the metaphyseal perichondrium is designated by a white arrow; and the scale bars equal 100 microns.

Fig 3: Identification of genes that are differentially expressed in superficial versus deep zone articular chondrocytes.a Lubricin (brown) is expressed in superficial zone chondrocytes (SZCs; adjacent to red bar) and not in deep zone chondrocytes (DZCs; adjacent to green bar) in bovine articular cartilage. Image of lubricin immunostaining taken from44. b Relative expression of genes that are differentially expressed in superficial zone bovine articular chondrocytes (SZC) versus deep zone bovine articular chondrocytes (DZC). Maximal and minimal expression levels in SZCs and DZCs are indicated by the intensity of the red and green hues, respectively. c Volcano plot of differentially expressed genes (DEGs) in SZCs and DZCs. Each dot represents one gene. The red dots represent SZC-specific DEGs, the green dots represent DZC-specific DEGs. Note that the TF gene Creb5 was as differentially expressed as Prg4, with ~25-fold higher levels in SZCs than in DZCs.

Fig 4: Creb5-dependent induction of Prg4 requires SAPK activity, but neither phosphorylation of T59 nor T61 in Creb5.a TGF-ß2 induced expression of Prg4 in superficial zone articular chondrocytes is blocked by inhibition of p38 activity. SZCs were cultured in either the absence (white) or presence (gray) of TGF-ß2 (20 ng/ml), plus a p38 inhibitor (SB203580; 10 µM), or a JNK inhibitor (SP600125; 10 µM), as indicated for 2 days. Gene expression was assayed by RT-qPCR and normalized to Gapdh. Error bar indicates standard error of the mean (n = 4 technical repeats for Prg4 expression, and n = 2 technical repeats for Creb5 expression). Similar results have been obtained in three independent biological repeats. b, c DZCs were infected with a lentivirus encoding doxycycline-inducible Creb5-HA. After selection in G418, the cells (iCreb5-DZCs) were cultured in either the absence or presence of doxycycline (1 µg/ml), TGF-ß2 (20 ng/ml), a p38 inhibitor (SB203580; 10 µM), or a JNK inhibitor (SP600125; 10 µM), as indicated for 2 days. b Gene expression was assayed by RT-qPCR and normalized to Gapdh. Error bar indicates standard error of the mean (n = 2 technical repeats). Similar results have been obtained in three independent biological repeats. c Western analysis of exogenous Creb5-HA expression and phosphorylation in iCreb5-DZCs with antibodies directed against either phospho-Creb5 (T61), total Creb5, HA, or a-tubulin. Similar results have been obtained in two independent biological repeats. d, e DZCs were infected with a lentivirus encoding either iCreb5-WT-HA; iCreb5-T59,61A-HA; or iCreb5-T59,61D-HA. After selection in G418, the cells were cultured in either the absence or presence of doxycycline (1 µg/ml), TGF-ß2 (20 ng/ml), and TGF-a (100 ng/ml) as indicated for 3 days. d Western analysis of iCreb5-WT/mutant expression in iCreb5-DZCs with antibodies directed against either phospho-Creb5 (T61) or total Creb5. Similar results have been obtained in two independent biological repeats. e Gene expression was assayed by RT-qPCR and normalized to Gapdh. Error bar indicates standard error of the mean (n = 2 technical repeats). Similar results have been obtained in three independent biological repeats.

Fig 5: Transcription factor binding sites that are enriched in ATAC-Seq peaks which display differential accessibility in superficial versus deep zone chondrocytes.a Homer motif analysis of ATAC-Seq peaks that are enriched in SZCs versus DZCs indicated that binding sites for Creb5, Nfi, Nfat, and Tbx TFs are enriched in superficial zone-specific ATAC-Seq peaks. b Homer motif analysis of ATAC-Seq peaks that are enriched in DZCs versus SZCs indicated that the binding sites for Tead and Runx TFs are enriched in deep zone-specific ATAC-Seq peaks. In both (a, b), the frequency of these sequence motifs in zone-specific ATAC-Seq peaks (% of targets) versus their frequency in the genome (% background) is displayed. c Correlation between ATAC-Seq sites and zone-specific gene expression. A high correlation was observed between zone-specific ATAC-Seq sites (located = 25 kb from the transcription start site) and zone-specific gene expression in both SZCs and DZCs. This correlation was highest at the Prg4 locus, where four distinct regions (E1–E4) were selectively accessible in SZCs.