1. Volume 27, Issue 1, Pages 53-66 (July 2007)
XBP1 Controls Diverse Cell Type- and Condition-Specific Transcriptional Regulatory Networks 
Diego Acosta-Alvear, Yiming Zhou, Alexandre Blais, Mary Tsikitis, Nathan H. Lents, Carolina Arias, Christen J. Lennon, Yuval Kluger, Brian David Dynlacht 
Molecular Cell 
Volume 27, Issue 1, Pages (July 2007)
DOI: /j.molcel
Copyright © 2007 Elsevier Inc. Terms and Conditions

2. Figure 1 Genome-wide Identification of XBP1 Targets
(A) Strategy for the identification of XBP1 targets in various cell types.
(B) RT-PCR analyses showing the relative levels of XBP1-u and XBP1-s in the five cell populations studied. Xbp1, both splice forms; Xbp1-s, spliced message only. Gapdh, loading control; Unt., untreated cells.
(C) ChIP assay for XBP1 in myotubes treated with IGF1. Input, PCR reactions performed with 0.5% or 0.25% of total starting material.
(D) XBP1 ChIP in myotubes treated with 2DG. Inputs as in (C).
(E) Heat map showing 407 XBP1-bound promoter regions in skeletal muscle, plasma, or pancreatic β cells. Scores from 0–3 that reflect increasing confidence levels in a given binding event were assigned to each bound locus (Supplemental Experimental Procedures). Loci bound in two or three replicates within the same biological condition were assigned a corresponding score. Loci bound in a single experiment in any particular condition that were also bound in at least two replicates in any other condition were assigned a score of < 2. Loci bound in a single experiment were considered not bound and were given a score of 0.
(F) Venn diagrams showing overlap between genes bound by XBP1 in different cell populations. “ER-stressed skeletal muscle,” genes bound in myotubes treated with 2DG or IGF1; “dedicated secretory cells,” genes bound in plasma or pancreatic β cells.
(G) ChIP assay for XBP1 in mouse skeletal muscle tissue. Input, PCR reactions performed with 0.2% or 0.1% of total starting material.
Molecular Cell  , 53-66DOI: ( /j.molcel )
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3. Figure 2 Validation of Cell Type- and Condition-Specific XBP1 Targets
(A) ChIP assay for XBP1 in diverse conditions and cell types. Gene ontology (GO) categories are indicated for three clusters of XBP1 targets (Table S3). Input, PCR reactions performed with 0.2% or 0.1% of total starting material.
(B) ChIP assay for XBP1 targets selectively bound in different cell types. Input, PCR reactions performed with 0.2%, 0.1%, or 0.05% of total starting material.
Molecular Cell  , 53-66DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

4. Figure 3
XBP1 Binds Targets Involved in Unexpected Functional Categories
(A) Distribution of manually curated GO annotations for XBP1 targets. In some instances, a gene is assigned to more than one category. The percentage refers to the number of bound genes within any particular GO category in relation to the total number of bound genes that have a GO annotation (N = 345). The histogram depicts the distribution of GO categories among different cell types as well as the percentage of bound genes within each group. Percentages for the histogram were calculated as described above.
(B) XBP1 binds the promoters of genes involved in amyloid precursor protein (APP) trafficking and processing. XBP1-target interactions as revealed by ChIP-on-chip are shown with black arrows, and published interactions are shown with blue edges. Connections between APP, IBM, and AD are shown in red.
(C) ChIP assay for selected XBP1 targets that are components of the γ-secretase complex or modulators of APP vesicular trafficking. Input, PCR reactions performed with 0.2% or 0.1% of total starting material.
Molecular Cell  , 53-66DOI: ( /j.molcel )
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5. Figure 4 Definition of Physiological XBP1 Binding Sites
(A) Sequence logos and consensus cis-regulatory elements associated with PWMs discovered in XBP1 target promoters. Enrichment p values were calculated based on the hypergeometric probability.
(B) Frequency distribution plot for the XBP1 PWMs identified by de novo approaches and the reconstructed ERSE and ERSE-II among experimentally determined XBP1-bound sequences. All four possible topologies of the ERSE and ERSE-II were considered (D).
(C) Venn diagrams showing the number of experimentally identified genes that have matches for the indicated XBP1 PWMs. In contrast to (B), the diagrams consider only the canonical topologies of the ERSE and ERSE-II. The upper diagram shows the overlap between the genes that have significant MATCH scores for the “short” XBP1 PWMs shown in (A) and those that have the UPRE, ERSE, or ERSE-II. The lower diagram shows the overlap between genes that have matches for each of these individual motifs.
(D) Statistical analysis of the abundance of the topological permutations of the ERSE and ERSE-II among XBP1 targets based on hypergeometric probability. Green bars, proportion of unique bound genes that have a match for the indicated permutation of the ERSE or ERSE-II; gray bars, proportion of unique genes that have a match for the same permutation of the ERSE or ERSE-II among all genes on the microarray. Canonical and permutated topologies of the ERSE and ERSE-II are shown. Arrows, CAAT box (blue) or the CCACG box for XBP1 (red); arrows pointing to the right, motif on sense strand; arrows pointing to the left, motifs on antisense strand; solid boxes, 9 bp or 1 bp spacers.
Molecular Cell  , 53-66DOI: ( /j.molcel )
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6. Figure 5 XBP1 Binding Is Associated with Gene Activation
(A) Gene expression changes during myogenic differentiation and after ectopic expression of XBP1-s. Cell populations (columns) and individual genes (rows) are indicated. Profiles of myotubes, myoblasts, and myoblasts expressing XBP1-s were compared to control GFP-transduced myoblasts. Only genes with a 2-fold or greater difference in expression are depicted. XBP1 binding to target genes in stressed muscle cells is indicated by blue bars next to the heat map. DM, differentiation medium.
(B) RT-PCR validation of expression profiling results.
(C) Frequency distribution of genes according to their differential expression in plasma cells versus naive B cells. Genes bound by XBP1 in hybridoma cells are represented by the dark blue trace (irrespective of their GO annotation) or by the light blue trace (“unexpected” GO categories only); all others in the dataset are represented by the red trace. The enrichment in genes expressed at higher levels in plasma than naive B cells among XBP1 targets versus the whole dataset is calculated from the hypergeometric probability (Table S6).
(D) A distribution plot similar to the one shown in (C), comparing germinal centers and naive B cells.
(E and F) Frequency distribution of genes according to their differential expression in pancreatic islet and skeletal muscle tissues. The enrichment for the association between XBP1 binding and increased gene expression in pancreatic islets (E) or following tunicamycin treatment of fibroblasts (F) is calculated as in (C) (Table S6). In all cases, genes bound by XBP1 in a given cell type or under a certain condition are represented by the blue trace; all others are represented by the red trace.
Molecular Cell  , 53-66DOI: ( /j.molcel )
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7. Figure 6
Effects of Ectopic XBP1-s Expression and Acute ER Stress on Myogenic Differentiation and Mist1 Transcription
(A) Immunofluorescence (IF) analysis of C2C12 myogenic differentiation. Myoblasts were transduced with a retrovirus encoding XBP-s or a retrovirus encoding GFP as a control and induced to differentiate. After 4 days, myosin heavy chain (MyHC) expression was visualized. The panels on the right show a 2× magnification with respect to the panels on the left.
(B) RT-PCR detection of terminal differentiation markers in XBP-s- or GFP-transduced skeletal muscle cells. The cells were cultured as described in (A).
(C) ChIP validation of XBP1 binding to the Mist1 promoter in myoblasts and dedicated secretory cells. Input, PCR reactions performed with 0.2% of total starting material.
(D) RT-PCR detection of Xbp1 and Mist1 mRNA levels in skeletal muscle cells subjected to different types of ER stress, transduced with a retrovirus encoding XBP1-s, and/or transfected with XBP1 siRNA duplexes. ER stress was induced by thapsigargin (Tg), tunicamycin (Tm), or 2DG. The ER stress utilized in the right-hand panel was Tg in the mock- and RV-GFP-transduced cells and 2DG in the cells transfected with siRNAs.
Molecular Cell  , 53-66DOI: ( /j.molcel )
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8. Figure 7 Ectopic Expression of Mist1 Impairs Myogenic Differentiation
(A) Analysis of C2C12 myogenic differentiation. Myoblasts were transduced with retroviruses encoding Mist1 or GFP as a control, then induced to differentiate and processed as in Figure 6A.
(B) RT-PCR detection of terminal differentiation markers in Mist1- or GFP-transduced skeletal muscle cells. The cells were cultured as described for (A).
(C) Model in which the actions of XBP1 are linked to the differentiation of acinar pancreas or skeletal muscle (see text for details). Interactions between XBP1 and its targets are shown with black arrows. Published interactions are shown with dotted edges.
Molecular Cell  , 53-66DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions