Khetchoumian K , Balsalobre A , Mayran A , Christian H , Chénard V , St-Pierre J , Drouin J .

MOP-86650 CIHR, FRN-154297 CIHR

	Figure 1. Tpit is required for postnatal maturation of pituitary POMC cells. a–o Reduced cell size and organelle content in Tpit-deficient pituitaries. a, b Nuclear staining (Hoechst) of pituitary sections from adult WT a and KO b mice. Demarcations between pituitary lobes (anterior: AL, intermediate: IL, posterior: PL) are indicated by dashed lines. Higher magnification insets show increased nuclear density in mutant IL. Scale bars: 10 µm a, 20 µm b. c, d Quantitation of total genomic DNA c and RNA d contents in WT and Tpit KO IL (each dot represents independent measure). e–h Flow cytometry (FACS) analysis of WT e and Tpit-KO f IL cells showing forward (FSC) vs. side (SSC) scatter that reflect cell size and granularity (organelle content), respectively. Distribution of cell size g and granularity h for each genotype. i, j RNA content (histogram) and FACS analyses of POMC-deficient IL cells (purple) indicate normal RNA content, size i and slightly reduced granularity j. k–o Ultrastructure analyses confirm cell size and organelle content defects in Tpit-deficient IL. k, l Electron micrographs of sections from WT k or KO l adult mouse pituitaries. WT cells k are rounded (dashed line), contain dense-cored secretory granules (g), mitochondria (m), and some rough endoplasmic reticulum (RER). Mutant cells l are smaller, with little cytoplasm and organelles (mitochondria dominate) and have a stellate appearance (not rounded). Scale bar in k = 2 µm, in l = 1 µm. n = nucleus. m–o Quantitation of cell area m, RER content n and granule density o in WT or KO IL cells. Data are presented as means (n = 4 mice) ± SEM. p–r Pituitary POMC cells develop into secretory factories postnatally. p Melanotrope cell size changes after birth. FACS profiles showing FSC distribution of IL GFP-positive cells from POMC-EGFP mice. Numbers indicate calculated cell volumes (µm3 x 10−3). q, r Summary of size q and granularity/organelle content r changes in postnatal IL melanotropes (filled circles) and AL corticotropes (empty circles). Inferred progression of cell size and granularity in melanotropes (blue) and corticotropes (green) between days P1 and P90 (adult). Size and granularity of Tpit-KO cells remain at the P1 stage (red). Compared to controls using bilateral Student’s T-test with unequal variances: *P < 0.05, **P < 0.005, ***P < 0.0005
	Figure 2. Tpit controls expression of translation and secretory pathway genes. a–e Tpit transcriptional signature in the pituitary. a Tpit-dependent genes identified by comparison of WT and KO IL transcriptomes. b Gene ontology (GO) distribution of transcripts downregulated in Tpit-deficient IL (yellow compared to random in gray) showing enrichment of protein transport, secretory pathway, and translation genes. BP biological process. CC cellular component. P-values relative to random occurrence using modified Fisher Exact P-value (EASE scores provided by DAVID) are shown. c Heat map representation (log2 of changes relative to median) of expression for a subset of genes in two WT and KO ILs. Each column represents an IL sample, and each row a gene. Blue and yellow represent up- and down- regulation, respectively. GTFs general transcription factors. d Real-time quantitative PCR (RT-qPCR) validation of expression changes for a subset of UPR pathway genes in Tpit-KO (red) and POMC-KO ILs (purple). Relative mRNA levels were normalized to TBP mRNA and represented as fraction of WT levels (black). Means ± SEM of 3–6 mice per genotype (each dot represents independent measure). e Postnatal expression of Tpit, POMC, Creb3l2, and UPR pathway genes in melanotropes. Relative IL mRNA levels (means ± SEM of 5–8 animals per group) measured by RT-qPCR were normalized to TBP and represented as fraction of P1 levels. f Loss of Creb3l2 expression in Tpit KO IL assessed by Creb3l2 immunofluorescence on P17 pituitary sections. g Western blot analysis of Creb3l2 in WT and Tpit KO ILs, and in AtT-20 cells. FL, full-length, CL, cleaved, NS, non specific. h, i Creb3l2 is a major Tpit target. h Top: ChIPseq profiles in AtT-20 cells revealing direct recruitment of Tpit to a Creb3l2 intron 1 sequence exhibiting active enhancer marks, namely bimodal H3K4me1, H3K27ac and ATAC peaks. ATACseq profiles in FACS-purified normal mouse melanotropes (MSH) and corticotropes (ACTH) are shown for comparison. Bottom: two conserved Tpit-RE palindromic sequences at the Tpit recruitment region of Creb3l2 intron 1. i Dose-dependent activation by Tpit of Luciferase reporter containing the intron1 enhancer (986 bp) of the Creb3l2 gene assessed by transfection into Tpit-negative αT3 pituitary cells. Compared to controls using bilateral Student’s T-test with unequal variances: *P < 0.05, **P < 0.005, ***P < 0.0005
	Figure 3. Creb3l2 and XBP1 regulate different aspects of secretory capacity. a Schematic view of dominant negative inhibition of bZIP TFs by AZIP proteins (adapted from ref. 28). Left: free bZIP/bZIP homodimer with unstructured basic region (blue). Middle: DNA bound bZIP/bZIP homodimer with α-helical basic region. Right: bZIP/AZIP heterodimer where the basic region and the designed acidic amphipathic extension (red) interact as α-helices to extend the coiled-coil domain and prevent DNA binding. b Efficiency of overexpressed (left) or endogenous (right) Creb3l2 inhibition by ACreb3l2. The Creb3l2-RE Luciferase reporter (3xCreb3l2-RE-luc) was used to assess Creb3l2 activity and ACreb3l2 antagonism upon transfection into INS-1 cells; similar results were obtained in AtT-20 cells. c Total IL RNA contents measured in WT, ACreb3l2, AXBP1, ACreb3l2/AXBP1 transgenic, and Tpit-KO mice. Data are from 6 to 19 pools of 5–10 ILs. d, e Melanotrope nuclear density (Tpit nuclear staining: red) on histological sections of ILs from WT, ACreb3l2, AXBP1, and ACreb3l2/AXBP1 transgenic mice d and their quantifications in arbitrary units (AU) relative to WT melanotrope density adjusted to 1 e. f–h Creb3l2 and XBP1 gain of function in stably infected AtT-20 cells. FACS analyses showing cell size f and granularity g index for AtT-20 cells expressing Creb3l2, XBP1, or both compared to control neomycin-resistant (Neo) AtT-20 cells. Data (±SEM, n = 3) are presented relative to Neo cells set to 1. h Total RNA content of control AtT-20 cells (Neo) and AtT-20 cells expressing Creb3l2, XBP1 or both (±SEM, n = 3). i–l Relative contributions of Creb3l2 and XBP1 to secretory capacity. i SUnSET (puromycin incorporation) measurements of protein synthesis in IL cells of control (WT), ACreb3l2, AXBP1, ACreb3l2/AXBP1 transgenic, and Tpit-KO mice. Data in AU are presented as fractions of WT IL translation rates set to 1. j Melanotrope cell ER contents (ER-tracker) measured in WT, ACreb3l2, AXBP1, ACreb3l2/AXBP1 transgenic, and Tpit-KO mice. Data are presented as fraction of WT IL ER contents set to 1. k Total protein content of control AtT-20 cells (Neo) and AtT-20 cells expressing Creb3l2, XBP1, or both. l CRH-induced ACTH release from the indicated pools of AtT-20 cells. Each dot represents independent measure. Compared to controls using bilateral Student’s T-test with unequal variances: * P < 0.05, **P < 0.005, ***P < 0.0005
	Figure 4. Creb3l2 targets the promoters of translation genes. a–d ChIPseq profiles for Creb3l2 and XBP1 at regulatory sequences of ER a and translation b–d genes. ChIPseq patterns are shown for control IgG and FLAG, Creb3l2, XBP1, H3K4me1, and ATACseq. ATACseq profiles in FACS-purified normal mouse melanotropes (MSH) and corticotropes (ACTH) are shown for comparison. e GO terms of genes associated with TSS proximal (<1 kb) Creb3l2 peaks. Peaks were assigned to the closest gene with the AnnotatePeaks Homer command. Bars represent the number of genes in each category associated with Creb3l2 peaks (green) or random occurrence of genes in each category (gray). P-values relative to random occurrence using hypergeometric distribution provided by AmiGO are shown above the bars. ns not significant. The bottom panels provide boxplot representation of the distance to TSS for Creb3l2 peaks of each GO category revealing promoter-proximal associations for groups with significant associations. Center lines show medians; box limits indicate the 25th and 75th percentiles; whiskers extend to 1.5 times the interquartile range from the 25th to 75th percentiles. f Dose-dependent activation by Creb3l2 of luciferase reporter containing three copies of Creb3l2-binding sites (3xCreb3l2-RE) upon transfection into pituitary GH3 cells. g Creb3l2 binds 70–80% of the promoters of translation genes. Pie charts showing proportion of genes (absolute gene numbers indicated between parentheses and target genes listed in Supplementary Table 2) bound by Creb3l2 (green) in each category related to translation, namely: translation factors, ribosomal proteins, and aminoacyl tRNA synthetases. The spermatogenesis group serves as control. Gene lists for each category are from MGI (http://www.informatics.jax.org/mgihome/GO/project.shtml)
	Figure 5. Creb3l2 is a scaling factor for translation. a Tpit KO transcriptome signature is largely phenocopied by inhibition of Creb3l2 and XBP1. Heatmap representation of gene clustering identified by global analysis of expression profiling datasets (left) and GO distribution of genes in each cluster (right). Transcriptome data from WT, Tpit-KO, ACreb3l2, AXBP1, and ACreb3l2/AXBP1 transgenic mice and from the gain-of-function AtT-20 cells were clustered with the Cluster 3.0 software using K-means. P-values relative to random occurrence using modified Fisher Exact P-value (EASE scores provided by DAVID) are shown. b Decreased lactate dehydrogenase (Ldha) and pyruvate dehydrogenase kinase (Pdk1) mRNAs (RNAseq normalized counts) in AtT-20 cells expressing Creb3l2 + XBP1 relative to control (Neo) cells set to 1. c Increased ATP turnover, decreased lactate production and glucose utilization, showing shift of ATP production from glycolysis to oxidative phosphorylation in AtT-20 cells expressing Creb3l2 + XBP1. Data represent the means ± SEM (n = 5–6). d Adaptation (5 weeks) of Xenopus skin color to black (right) background compared to white (left) increases total IL RNA content (ng ± SEM, for n = 4–5). e Expression of POMC and UPR genes in white (white bars) and black (black bars) background adapted frogs. Relative IL mRNA levels measured by RT-qPCR were normalized to those of β-actin and represented as fraction of levels in white background set to 1. Data represent means ± SEM (each dot represents independent measure). f–h Expression of Creb3l2 in INS-1 cells. FACS analyses of cell size f and granularity g index for INS-1 cells expressing Creb3l2. All data represent the means ± SEM (n = 3). h SUnSET measurement of protein synthesis in control INS-1 cells (Neo) and INS-1 cells expressing Creb3l2. Data (±SEM, n = 3) are presented relative to Neo cell set to 1. Compared to controls using bilateral Student’s T-test with unequal variances: *P < 0.05, **P < 0.005, ***P < 0.0005
	Figure 6. Developmental mechanism to establish high capacity secretory cells. Freshly differentiated pituitary cells have basic secretory features and acquire the features of hormone-producing factories during early post-natal development under the concerted actions of two bZIP transcription factors Creb3l2 and XBP1. This is controlled by the determination transcription factor Tpit in pituitary POMC cells (likely PDX1 in pancreatic β cells and BLIMP1 in activated B cells). a In pituitary, Tpit activates expression of Creb3l2 and XBP1 by directly targeting their genes. b Both factors require activation through either proteolytic cleavage (Creb3l2) or alternative splicing (XBP1) and this appears to be either cell-autonomous in normal development or may be regulated by developmental signals as shown for TGFβ in liver23. c Translation capacity and protein transport are enhanced by the scaling action of Creb3l2 directly targeting the promoters of a large number of genes involved in translation including genes for regulatory factors, ribosomal proteins, and tRNA aminoacyl transferases. In parallel, XBP1 stimulates ER biogenesis without triggering the protein stress response, thus activating the physiological UPR response
	Supplementary Figure 1. Validation of Tpit KO (Tpit-/-) transcriptome data. (a-c) Scatter plot comparisons of 2 WT (a) and 2 KO (b) pituitary intermediate lobe (IL) transcriptomes showing the reproducibility of microarray experiments and comparison of WT vs Tpit KO pituitary IL transcriptomes (c). (d) Expression (Affymetrix microarray signals) of translation regulators, ERAD and apoptotic genes in WT (black bars) and KO (red bars) ILs. P-values determined using local-pooled-error (LPE) test. Compared to controls (WT): ** P< 0.005, *** P< 0.0005. (e) List of 10 most downregulated transcription factor genes in Tpit-deficient ILs. (f) Expression of the OASIS family members, Tpit and POMC in different tissues and cell lines.
	Supplementary Figure 2. Efficient and specific inhibition of Creb3l2 and XBP1 activity using acidic dominant-negative constructs (AZIP). (a) Expression (Flag Western blot) of FL-Creb3l2 in AtT-20 cells results in production of both FL and cleaved (CL) active forms of Creb3l2. Treatment with the proteasome inhibitor MG132 leads to stabilization of Creb3l2. (b) Inhibition of Creb3l2, but not XBP1, activity by overexpression of ACreb3l2. (c) Inhibition of XBP1, but not Creb3l2, activity by overexpression of AXBP1. The 3xCreb3l2/XBP1-RE Luciferase reporter was used to assess Creb3l2 or XBP1 activity upon transfection into INS-1 cells. (d-e) RT-qPCR quantification of ACreb3l2 and AXBP1 transcripts relative to the endogenous Creb3l2 and XBP1s transcripts in ILs of the transgenic mice reported in Fig. 3. (f) RT-qPCR assessment of rRNA 18S and 28S contents in WT, LOF transgenics and Tpit KO ILs. Data represent the average ±SEM of 4 pools of 5-10 ILs. Statistical significance was assessed using bilateral Student’s T-test with unequal variances. ** P<0.005, *** P<0.0005. (g) Enrichment of translation genes in transcriptomes of plasmablasts treated for 72h with the selective S1P (S1 protease responsible for Creb3l2 cleavage) inhibitor PF-429242 (analysis of raw data from Al-Maskari et al. 2). (h) mTORC1 status is not affected by Tpit KO or Creb3l2/XBP1 downregulation (ACreb3l2, AXBP1). Western blot showing similar active mTOR (phospho Ser-2481) to total mTOR levels in IL protein extracts from indicated mutant mice.
	Supplementary Figure 3. Creb3l2 and XBP1 target the promoters of translation and ER biogenesis genes, respectively. (a-c) Genomic distribution of Tpit, Creb3l2 and XBP1 ChIPseq peaks. (d-f) ChIP-qPCR validation of Creb3l2 ChIPseq. Data for 8 representative promoter loci are shown as assessed by FLAG antibody ChIP-qPCR (d), FLAG antibody ChIPseq (e) and Creb3l2 antibody ChIP-qPCR (f). (g) GO terms of genes associated with TSS proximal (≤1 kb) XBP1 peaks. Peaks were assigned to the closest gene with the AnnotatePeaks Homer command. Bars represent the number of genes in each category associated with XBP1 peaks (blue) or random occurrence of genes in each category (grey). P-values relative to random occurrence determined by using hypergeometric distribution provided by AmiGO are shown above the bars; ns, not significant. The bottom panels provide boxplot representation of the distance to TSS for XBP1 peaks of each GO category revealing promoter-proximal associations for groups with significant associations. Center lines show medians; box limits indicate the twenty-fifth and seventy-fifth percentiles; whiskers extend to 1.5 times the interquartile range from the twenty-fifth to seventy-fifth percentiles. BP: biological process, CC: cellular component. (h-k) ChIPseq profiles at regulatory sequences of genes targeted by Creb3l2 and/or XBP1. ChIPseq patterns are shown for control IgG, Flag, Creb3l2, XBP1, H3K4me1 and ATACseq. (l,m) Average profiles of H3K4me1 ChIPseq and ATACseq at Creb3l2 (l) or XBP1 (m) peaks.
	Supplementary Figure 4. Creb3l2 and XBP1 activate transcription through binding of a similar sequence motif. (a-b) Creb3l2-RE and XBP1-RE consensus identified by de novo motif searches in sequences bound by Creb3l2 and XBP1. (c-d) The transcriptional activity of Creb3l2 and XBP1 assessed by transfection in GH3 cells using two pGL4.10-luciferase reporters. Schematic representation of Luciferase reporters containing the Ssr3 promoter (c) or Kcnma1 enhancer (d) and dose-response curves. (e) ChIPseq profiles of Pdx1 at regulatory sequences of Creb3l2 in pancreatic beta cells. Data from http://chip-atlas.org (the Pdx cistrome of pancreatic islets, ERX103428, ERX103429). (f) RT-qPCR validation of RNAseq analyses at 8 representative loci. Statistical significance was assessed using bilateral Student’s T-test with unequal variances. * P<0.05, ** P<0.005, *** P<0.0005 compared to WT.
	Supplemenrtary Figure 5. Gating strategy used in FACS experiments. Followed IL dissociation cells are stained with propidium iodide (PI) and analyzed by FACS Calibur cell sorter (BD BioSciences). Gating and data analysis are done using the Summit 4.3 software. (a) A first gate (R1) is drawn to exclude cell debris and doublets or cell aggregates. (b) PI-positive (dead) cells are then excluded from the R1 gate, giving the R2 population. (c) Cells from R1 & R2 gates (single, live cells) are analyzed for different properties (FSC, reflecting cell size in the given example).
	Fig. 1. Tpit is required for postnatal maturation of pituitary POMC cells. a–o Reduced cell size and organelle content in Tpit-deficient pituitaries. a, b Nuclear staining (Hoechst) of pituitary sections from adult WT a and KO b mice. Demarcations between pituitary lobes (anterior: AL, intermediate: IL, posterior: PL) are indicated by dashed lines. Higher magnification insets show increased nuclear density in mutant IL. Scale bars: 10 µm a, 20 µm b. c, d Quantitation of total genomic DNA c and RNA d contents in WT and Tpit KO IL (each dot represents independent measure). e–h Flow cytometry (FACS) analysis of WT e and Tpit-KO f IL cells showing forward (FSC) vs. side (SSC) scatter that reflect cell size and granularity (organelle content), respectively. Distribution of cell size g and granularity h for each genotype. i, j RNA content (histogram) and FACS analyses of POMC-deficient IL cells (purple) indicate normal RNA content, size i and slightly reduced granularity j. k–o Ultrastructure analyses confirm cell size and organelle content defects in Tpit-deficient IL. k, l Electron micrographs of sections from WT k or KO l adult mouse pituitaries. WT cells k are rounded (dashed line), contain dense-cored secretory granules (g), mitochondria (m), and some rough endoplasmic reticulum (RER). Mutant cells l are smaller, with little cytoplasm and organelles (mitochondria dominate) and have a stellate appearance (not rounded). Scale bar in k = 2 µm, in l = 1 µm. n = nucleus. m–o Quantitation of cell area m, RER content n and granule density o in WT or KO IL cells. Data are presented as means (n = 4 mice) ± SEM. p–r Pituitary POMC cells develop into secretory factories postnatally. p Melanotrope cell size changes after birth. FACS profiles showing FSC distribution of IL GFP-positive cells from POMC-EGFP mice. Numbers indicate calculated cell volumes (µm3 x 10−3). q, r Summary of size q and granularity/organelle content r changes in postnatal IL melanotropes (filled circles) and AL corticotropes (empty circles). Inferred progression of cell size and granularity in melanotropes (blue) and corticotropes (green) between days P1 and P90 (adult). Size and granularity of Tpit-KO cells remain at the P1 stage (red). Compared to controls using bilateral Student’s T-test with unequal variances: *P < 0.05, **P < 0.005, ***P < 0.0005
	Fig. 2. Tpit controls expression of translation and secretory pathway genes. a–e Tpit transcriptional signature in the pituitary. a Tpit-dependent genes identified by comparison of WT and KO IL transcriptomes. b Gene ontology (GO) distribution of transcripts downregulated in Tpit-deficient IL (yellow compared to random in gray) showing enrichment of protein transport, secretory pathway, and translation genes. BP biological process. CC cellular component. P-values relative to random occurrence using modified Fisher Exact P-value (EASE scores provided by DAVID) are shown. c Heat map representation (log2 of changes relative to median) of expression for a subset of genes in two WT and KO ILs. Each column represents an IL sample, and each row a gene. Blue and yellow represent up- and down- regulation, respectively. GTFs general transcription factors. d Real-time quantitative PCR (RT-qPCR) validation of expression changes for a subset of UPR pathway genes in Tpit-KO (red) and POMC-KO ILs (purple). Relative mRNA levels were normalized to TBP mRNA and represented as fraction of WT levels (black). Means ± SEM of 3–6 mice per genotype (each dot represents independent measure). e Postnatal expression of Tpit, POMC, Creb3l2, and UPR pathway genes in melanotropes. Relative IL mRNA levels (means ± SEM of 5–8 animals per group) measured by RT-qPCR were normalized to TBP and represented as fraction of P1 levels. f Loss of Creb3l2 expression in Tpit KO IL assessed by Creb3l2 immunofluorescence on P17 pituitary sections. g Western blot analysis of Creb3l2 in WT and Tpit KO ILs, and in AtT-20 cells. FL, full-length, CL, cleaved, NS, non specific. h, i Creb3l2 is a major Tpit target. h Top: ChIPseq profiles in AtT-20 cells revealing direct recruitment of Tpit to a Creb3l2 intron 1 sequence exhibiting active enhancer marks, namely bimodal H3K4me1, H3K27ac and ATAC peaks. ATACseq profiles in FACS-purified normal mouse melanotropes (MSH) and corticotropes (ACTH) are shown for comparison. Bottom: two conserved Tpit-RE palindromic sequences at the Tpit recruitment region of Creb3l2 intron 1. i Dose-dependent activation by Tpit of Luciferase reporter containing the intron1 enhancer (986 bp) of the Creb3l2 gene assessed by transfection into Tpit-negative αT3 pituitary cells. Compared to controls using bilateral Student’s T-test with unequal variances: *P < 0.05, **P < 0.005, ***P < 0.0005
	Fig. 3. Creb3l2 and XBP1 regulate different aspects of secretory capacity. a Schematic view of dominant negative inhibition of bZIP TFs by AZIP proteins (adapted from ref. 28). Left: free bZIP/bZIP homodimer with unstructured basic region (blue). Middle: DNA bound bZIP/bZIP homodimer with α-helical basic region. Right: bZIP/AZIP heterodimer where the basic region and the designed acidic amphipathic extension (red) interact as α-helices to extend the coiled-coil domain and prevent DNA binding. b Efficiency of overexpressed (left) or endogenous (right) Creb3l2 inhibition by ACreb3l2. The Creb3l2-RE Luciferase reporter (3xCreb3l2-RE-luc) was used to assess Creb3l2 activity and ACreb3l2 antagonism upon transfection into INS-1 cells; similar results were obtained in AtT-20 cells. c Total IL RNA contents measured in WT, ACreb3l2, AXBP1, ACreb3l2/AXBP1 transgenic, and Tpit-KO mice. Data are from 6 to 19 pools of 5–10 ILs. d, e Melanotrope nuclear density (Tpit nuclear staining: red) on histological sections of ILs from WT, ACreb3l2, AXBP1, and ACreb3l2/AXBP1 transgenic mice d and their quantifications in arbitrary units (AU) relative to WT melanotrope density adjusted to 1 e. f–h Creb3l2 and XBP1 gain of function in stably infected AtT-20 cells. FACS analyses showing cell size f and granularity g index for AtT-20 cells expressing Creb3l2, XBP1, or both compared to control neomycin-resistant (Neo) AtT-20 cells. Data (±SEM, n = 3) are presented relative to Neo cells set to 1. h Total RNA content of control AtT-20 cells (Neo) and AtT-20 cells expressing Creb3l2, XBP1 or both (±SEM, n = 3). i–l Relative contributions of Creb3l2 and XBP1 to secretory capacity. i SUnSET (puromycin incorporation) measurements of protein synthesis in IL cells of control (WT), ACreb3l2, AXBP1, ACreb3l2/AXBP1 transgenic, and Tpit-KO mice. Data in AU are presented as fractions of WT IL translation rates set to 1. j Melanotrope cell ER contents (ER-tracker) measured in WT, ACreb3l2, AXBP1, ACreb3l2/AXBP1 transgenic, and Tpit-KO mice. Data are presented as fraction of WT IL ER contents set to 1. k Total protein content of control AtT-20 cells (Neo) and AtT-20 cells expressing Creb3l2, XBP1, or both. l CRH-induced ACTH release from the indicated pools of AtT-20 cells. Each dot represents independent measure. Compared to controls using bilateral Student’s T-test with unequal variances: * P < 0.05, **P < 0.005, ***P < 0.0005
	Fig. 4. Creb3l2 targets the promoters of translation genes. a–d ChIPseq profiles for Creb3l2 and XBP1 at regulatory sequences of ER a and translation b–d genes. ChIPseq patterns are shown for control IgG and FLAG, Creb3l2, XBP1, H3K4me1, and ATACseq. ATACseq profiles in FACS-purified normal mouse melanotropes (MSH) and corticotropes (ACTH) are shown for comparison. e GO terms of genes associated with TSS proximal (<1 kb) Creb3l2 peaks. Peaks were assigned to the closest gene with the AnnotatePeaks Homer command. Bars represent the number of genes in each category associated with Creb3l2 peaks (green) or random occurrence of genes in each category (gray). P-values relative to random occurrence using hypergeometric distribution provided by AmiGO are shown above the bars. ns not significant. The bottom panels provide boxplot representation of the distance to TSS for Creb3l2 peaks of each GO category revealing promoter-proximal associations for groups with significant associations. Center lines show medians; box limits indicate the 25th and 75th percentiles; whiskers extend to 1.5 times the interquartile range from the 25th to 75th percentiles. f Dose-dependent activation by Creb3l2 of luciferase reporter containing three copies of Creb3l2-binding sites (3xCreb3l2-RE) upon transfection into pituitary GH3 cells. g Creb3l2 binds 70–80% of the promoters of translation genes. Pie charts showing proportion of genes (absolute gene numbers indicated between parentheses and target genes listed in Supplementary Table 2) bound by Creb3l2 (green) in each category related to translation, namely: translation factors, ribosomal proteins, and aminoacyl tRNA synthetases. The spermatogenesis group serves as control. Gene lists for each category are from MGI (http://www.informatics.jax.org/mgihome/GO/project.shtml)
	Fig. 5. Creb3l2 is a scaling factor for translation. a Tpit KO transcriptome signature is largely phenocopied by inhibition of Creb3l2 and XBP1. Heatmap representation of gene clustering identified by global analysis of expression profiling datasets (left) and GO distribution of genes in each cluster (right). Transcriptome data from WT, Tpit-KO, ACreb3l2, AXBP1, and ACreb3l2/AXBP1 transgenic mice and from the gain-of-function AtT-20 cells were clustered with the Cluster 3.0 software using K-means. P-values relative to random occurrence using modified Fisher Exact P-value (EASE scores provided by DAVID) are shown. b Decreased lactate dehydrogenase (Ldha) and pyruvate dehydrogenase kinase (Pdk1) mRNAs (RNAseq normalized counts) in AtT-20 cells expressing Creb3l2 + XBP1 relative to control (Neo) cells set to 1. c Increased ATP turnover, decreased lactate production and glucose utilization, showing shift of ATP production from glycolysis to oxidative phosphorylation in AtT-20 cells expressing Creb3l2 + XBP1. Data represent the means ± SEM (n = 5–6). d Adaptation (5 weeks) of Xenopus skin color to black (right) background compared to white (left) increases total IL RNA content (ng ± SEM, for n = 4–5). e Expression of POMC and UPR genes in white (white bars) and black (black bars) background adapted frogs. Relative IL mRNA levels measured by RT-qPCR were normalized to those of β-actin and represented as fraction of levels in white background set to 1. Data represent means ± SEM (each dot represents independent measure). f–h Expression of Creb3l2 in INS-1 cells. FACS analyses of cell size f and granularity g index for INS-1 cells expressing Creb3l2. All data represent the means ± SEM (n = 3). h SUnSET measurement of protein synthesis in control INS-1 cells (Neo) and INS-1 cells expressing Creb3l2. Data (±SEM, n = 3) are presented relative to Neo cell set to 1. Compared to controls using bilateral Student’s T-test with unequal variances: *P < 0.05, **P < 0.005, ***P < 0.0005
	Fig. 6. Developmental mechanism to establish high capacity secretory cells. Freshly differentiated pituitary cells have basic secretory features and acquire the features of hormone-producing factories during early post-natal development under the concerted actions of two bZIP transcription factors Creb3l2 and XBP1. This is controlled by the determination transcription factor Tpit in pituitary POMC cells (likely PDX1 in pancreatic β cells and BLIMP1 in activated B cells). a In pituitary, Tpit activates expression of Creb3l2 and XBP1 by directly targeting their genes. b Both factors require activation through either proteolytic cleavage (Creb3l2) or alternative splicing (XBP1) and this appears to be either cell-autonomous in normal development or may be regulated by developmental signals as shown for TGFβ in liver23. c Translation capacity and protein transport are enhanced by the scaling action of Creb3l2 directly targeting the promoters of a large number of genes involved in translation including genes for regulatory factors, ribosomal proteins, and tRNA aminoacyl transferases. In parallel, XBP1 stimulates ER biogenesis without triggering the protein stress response, thus activating the physiological UPR response

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