normal human b lymphoblastoid cell line gm12878  (ATCC)

Journal: Nature Genetics

Article Title: Noncoding mutations cause super-enhancer retargeting resulting in protein synthesis dysregulation during B cell lymphoma progression

doi: 10.1038/s41588-023-01561-1

Figure Lengend Snippet: a , b , Copy number changes, that is, losses and gains seen in the genomes of ( a ) FL and ( b ) DHL samples. The GISTIC plots show significant focal copy number variation (including recurrent CDKN2A / B loss, MDM2 gain and PAX5 / ZCCHC7 gain) occurring during transformation to DHL. c , Copy number and single-nucleotide variants involving the PAX5 / ZCCHC7 locus. Most cases demonstrate a gain of ZCCHC7 upon transformation to DHL (Fisher’s test P = 0.0023). TAD, CTCF, H3K4me1, H3K4me3 and H3K27ac ChIP–seq data overlap the PAX5 / ZCCHC7 locus. The area of copy number gain of PAX5 / ZCCHC7 mostly overlaps the entirety of the TAD. Transformation-associated aSHM at PAX5 is enriched in the first intron and overlaps with H3K4me3 and H3K27ac peaks (gray vertical box).

Article Snippet: SUDHL10 cells (CRL-2963) were purchased from the ATCC, cultured in the laboratory and modified through HDR to form the SUDHL10 PAX5 -TSS m ut cell line, as described elsewhere in this paper.

Techniques: Transformation Assay, ChIP-sequencing

Journal: Nature Genetics

Article Title: Noncoding mutations cause super-enhancer retargeting resulting in protein synthesis dysregulation during B cell lymphoma progression

doi: 10.1038/s41588-023-01561-1

Figure Lengend Snippet: a , Plots demonstrating percentages of mutations overlapping H3K27ac peaks (left, n = 35,770) and H3K4me3 peaks (right, n = 25,740) in DHL compared to FL. Each pair of points represents an H3K4me3/H3K27ac peak region. The 15 most frequently mutated peaks are highlighted and labeled with the names of nearby genes. The P values were calculated by two-sided Wilcoxon signed-rank test. b , c , In DTX1 ( b ) and BCL2 ( c ) transformation-associated aSHM overlaps H3K4me3 and H3K27ac sites and occurs near the boundaries of TADs. d , In the PAX5 / ZCCHC7 region, transformation-associated aSHM overlaps H3K4me3 and H3K27ac sites and occurs near the boundaries of the TAD. The increased read depth of the PAX5 / ZCCHC7 regions in P2.DHL is shown. e , Occurrence of SE mutations in compartment A versus compartment B in FL (left, n = 7) and DHL (right, n = 6) tumors with each pair of dots connected by a gray line representing one lymphoma sample. The P values were calculated using two-sided Wilcoxon signed-rank tests. The horizontal line in each group represents its mean value, and the corresponding vertical line represents its s.d. bar. f , Fraction of SE regions in compartment A versus compartment B. The bar plot shows size fraction of SE and non-SE in compartment A and compartment B, respectively. The overall total size of SEs across the whole genome is approximately 37 Mb; 31 Mb are located within compartment A and represent 2.6% of the overall compartment A region. g , Normalized and averaged somatic mutation density of FL and DHL in relation to TAD boundaries. Starting from the center of every TAD boundary (gray dashed line), the left side represents regions inside of TADs and the right side represents areas outside of TADs. Intra-TAD boundaries are not considered in this analysis.

Article Snippet: SUDHL10 cells (CRL-2963) were purchased from the ATCC, cultured in the laboratory and modified through HDR to form the SUDHL10 PAX5 -TSS m ut cell line, as described elsewhere in this paper.

Techniques: Labeling, Transformation Assay, Mutagenesis

Journal: Nature Genetics

Article Title: Noncoding mutations cause super-enhancer retargeting resulting in protein synthesis dysregulation during B cell lymphoma progression

doi: 10.1038/s41588-023-01561-1

Figure Lengend Snippet: (a) Overall frequency of micro-insertions (left) and micro-deletions (right) observed in paired follicular lymphoma and DHL samples from n = 6 patients. Each pair of dots connected by a grey line represents a pair of follicular lymphoma and DHL samples from a single patient. The mutational frequencies on the y-axis are expressed as percentage of micro-insertions or micro-deletions out of all mutations. The P -values were calculated by two-sided Wilcoxon signed rank tests. The boxplots display the 25 th and 75 th percentiles and the median of each group of data. The whiskers represent the highest and lowest values within 1.5 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\rm{\times }}$$\end{document} × the inter-quartile range. (b) The number of micro-indels overlapping H3K4me3 and H3K27ac intersecting regions in DHL compared to follicular lymphoma. Each pair of points represents an intersection segment of H3K4me3 peak and H3K27ac peak. All n = 21,283 segments were ranked in descending order by the sum of micro-indels observed in DHL. Five segments that harbor over three micro-indels are highlighted and labeled with nearby genes. The P -value was calculated by two-sided Wilcoxon signed-rank test. (c) Microdeletions and micro-insertions observed in the PAX5 -TSS2 region in three DHL tumors, two diffuse large B cell lymphoma (DLBCL) cell lines, as well as two primary DLBCL tumors. In IGV, WGS data from these seven samples show eight micro-deletions (blue arrows) and one micro-insertion (red arrow) in this region. (d) Mutational hotspots in the PAX5 / ZCCHC7 locus previously reported in chronic lymphocytic leukemia (CLL) (top panel), DLBCL (middle panel), and in the current DHL series (bottom panel). All p -values were calculated by two-sided Wilcoxon signed-rank test. (e) Hi-C overlap of the PAX5 / ZCCHC7 locus shows bidirectional transcription ( Dis3 C / C represents cells depleted of RNA exosome activity to stabilize bidirectional ncRNA expression), accumulation of non-B DNA associated DNA/RNA hybrids (in Dis3 C / C cells via DRIP-seq), H3K27ac peaks and AID target site identification via translocation capture sequencing (via HTGTS-seq). These observations demonstrate that processes relating to DNA targeting by AID occur at the PAX5 promoter in mouse B cells.

Article Snippet: SUDHL10 cells (CRL-2963) were purchased from the ATCC, cultured in the laboratory and modified through HDR to form the SUDHL10 PAX5 -TSS m ut cell line, as described elsewhere in this paper.

Techniques: Labeling, Hi-C, Activity Assay, Expressing, Translocation Assay, Sequencing

Journal: Nature Genetics

Article Title: Noncoding mutations cause super-enhancer retargeting resulting in protein synthesis dysregulation during B cell lymphoma progression

doi: 10.1038/s41588-023-01561-1

Figure Lengend Snippet: (a) Transcript reconstitution of PAX5 demonstrates two TSS, canonical TSS and PAX5 -TSS2. The distribution of H3K4me3, H3K27ac, and aSHM overlapping the two TSS is shown. (b) PAX5 -TSS2 hotspot mutations and deletions seen in four of the DHLs in this study (P1, P2, P6, P9) and in other studies . The most frequent mutations, that characterize the PAX5 -TSS2 mut in Fig. are marked by a red frame. (c) In the SUDHL10 cells, deletion of PAX5 -TSS2 (left) or incorporation of TSS2 mut (right) leads to marginally decreased expression of PAX5 mRNA but significantly increased expression of ZCCHC7 . Student’s 2-tailed T-test (paired) used for statistical analysis in both panels. (d) Mutation heatmap of 29 DLBCL cell lines (left panel) and 24 DLBCL primary tumors (right panel) at ZCCHC7 and PAX5 TSS2. Cell line WGS/RNAseq data were obtained from CCLE and SRA: PRJNA854968 . Tumor WGS/RNAseq data were obtained from dbGaP: phs000235.v20.p6 . Cell lines or tumors showing ZCCHC7 copy number gain, ZCCHC7 fusion, or mutation in any of four PAX5 -TSS2 hotspots (as shown in (b) ) are included in the ‘Mutant’ group. Others, displaying no alteration in these regions, compose the ‘WT’ group. All of the ZCCHC7 fusion events were detected via WGS or RNAseq data and summarized in Extended Fig. . All of the hotspot mutations were confirmed by checking corresponding WGS data or RNAseq data in IGV, with at least five supporting reads. Six primary tumors without WGS data but showing ZCCHC7 fusion or PAX5 -TSS2 hotspot mutation in RNAseq data were also included. ‘WT’ samples showing poor read coverage (depth < 10) at the four mutational hotspots were discarded. Z-score transformed RNA expression of ZCCHC7 and PAX5 are shown at the bottom. (e and f) RNA expression of ZCCHC7 and PAX5 in the ‘WT’ and ‘Mutant’ groups of DLBCL cell lines ( e ) and tumors ( f ) described in ( d ). P-values were calculated by two-sided Wilcoxon rank-sum test. Boxplots display the 25th and 75th percentiles and median of each group. Whiskers represent the highest and lowest values within 1.5 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\rm{\times }}$$\end{document} × the inter-quartile range.

Article Snippet: SUDHL10 cells (CRL-2963) were purchased from the ATCC, cultured in the laboratory and modified through HDR to form the SUDHL10 PAX5 -TSS m ut cell line, as described elsewhere in this paper.

Techniques: Expressing, Mutagenesis, Transformation Assay, RNA Expression

Journal: Nature Genetics

Article Title: Noncoding mutations cause super-enhancer retargeting resulting in protein synthesis dysregulation during B cell lymphoma progression

doi: 10.1038/s41588-023-01561-1

Figure Lengend Snippet: (a) Significant negative correlation between RNA expression of PAX5 and ZCCHC7 in 11 DLBCL cell lines. The P -value was calculated by two-sided Spearman’s correlation test. (b) Schematic of the pipeline used to identify potential targets of enhancer retargeting in the B cell lymphoma genome. Overall results of this analysis are shown in Fig. . The transcriptomic data from 11 DLBCL cell lines were reported by Morin et al. . The list of 367 enhancer retargeting (ER) gene pairs was previously reported by Oh et al. . (c) Top 10 potential enhancer retargeting gene pair candidates ranked by the pipeline and filtered by ‘Hypermutated’ criteria. The P-values were estimated for ranking consistently high (or low) on multiple lists (see Methods). (d) Comparison of Spearman’s correlation coefficient between predicted gene pairs experiencing enhancer retargeting and affected in opposite directions (n = 10,158) versus those affected in the same direction (n = 9,518). The P -value is calculated by two-sided Wilcoxon rank sum test. The boxplots display the 25 th and 75 th percentiles and the median of each group of data. The whiskers represent the highest and lowest values within 1.5 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\times$$\end{document} × the inter-quartile range. (e) Proximity of FAM102A and SLC25A25-AS1 and somatic mutations occurring in the promotor of FAM102A .

Article Snippet: SUDHL10 cells (CRL-2963) were purchased from the ATCC, cultured in the laboratory and modified through HDR to form the SUDHL10 PAX5 -TSS m ut cell line, as described elsewhere in this paper.

Techniques: RNA Expression, Comparison

Journal: Nature Genetics

Article Title: Noncoding mutations cause super-enhancer retargeting resulting in protein synthesis dysregulation during B cell lymphoma progression

doi: 10.1038/s41588-023-01561-1

Figure Lengend Snippet: (a) Four independent 4C-seq experiments measuring interactions between the ZCCHC7 promoter and intragenic enhancer regions adjacent to PAX5 . In Δ PAX5 -TSS2 SUDHL10 ( PAX5 -TSS2 deletion shown with black arrow), the PAX5 intragenic enhancer region interacts more strongly with the promoter of ZCCHC7 relative to cells with intact PAX5 -TSS2 (see thickness of black lines). (b) Comparison of 4C-seq data from PAX5 TSS2 WT versus PAX5 TSS2 del cells, demonstrating that similar regions of the ZCCHC7-PAX5 SE show increased interaction with the ZCCHC7 gene. (c) The normalized coverage of the interactions (site 1, site 2, site 3, and site 4) in (a and b) is shown in the bar graph. Error bars represent the standard deviations. Two-sided Welch’s t-test were used; n = 4 for interaction between site 1 and 4C bait 1, n = 2 for other interactions. (d) Sanger sequencing of chr9:37,026,299-37,026,327 in SUDHL10 PAX5 TSS mut cells. The two mutations are located at chr9:37,026,315-37,026,316 (as in Extended Data Fig. ). (e) Summary of PAX5 H3K4me3 region mutations acquired upon transformation to DHL. The weblogos show the classical AID mutational signature, C→T/G→A with WRC/GYW motif (W = A/T, R = A/G, and Y = T/C), that dominates the DHL-specific mutations within this 3kb region of PAX5 . Specifically, 11 C→T/G→A mutations out of 28 DHL-associated mutations in this region reflect the WRC/GYW motif (labeled in red).

Article Snippet: SUDHL10 cells (CRL-2963) were purchased from the ATCC, cultured in the laboratory and modified through HDR to form the SUDHL10 PAX5 -TSS m ut cell line, as described elsewhere in this paper.

Techniques: Comparison, Sequencing, Transformation Assay, Labeling

Journal: Nature Genetics

Article Title: Noncoding mutations cause super-enhancer retargeting resulting in protein synthesis dysregulation during B cell lymphoma progression

doi: 10.1038/s41588-023-01561-1

Figure Lengend Snippet: a , Distribution of Spearman’s correlation coefficient between RNA expression of two genes located within the same TAD. In total, 19,676 gene pairs were investigated by analyzing transcriptomic data of 11 DLBCL cell lines to identify candidates of ER. The ZCCHC7–PAX5 pair is highlighted. b , The 4C-seq experiments from the ZCCHC7 promoter measuring the interaction with H3K27ac + H3K4me3 + peaks, including those at the PAX5 promoter. In SUDHL10 cells where the PAX5 -TSS2 has been replaced with a mutation identified in DHL ‘chr9: 37,026,315–37,026,316 GC->AT’ (mutated region shown with orange arrow; SUDHL10 PAX5 -TSS2 mut ), the PAX5 intragenic enhancer regions (sites 1, 2 and 3) interact with the promoter of ZCCHC7 more strongly than in SUDHL10 cells without the PAX5 -TSS2 mutation (see thickness of black lines). c , Normalized coverage of the 4C interactions for each 4C-seq viewpoint is shown. The TSS2 mutant region does not overlap the ZCCHC7 interaction domain of the PAX5 promoter. Data are presented as mean values ± s.d. Two-sided Welch’s t test was used; n = 3 for each group. d , Tissue microarrays containing triplicate samples of 33 DLBCL and 9 DHL with controls including benign spleen, lymph node, tonsil and low-grade FL were stained by immunohistochemistry for ZCCHC7. ZCCHC7 was visualized predominantly in nucleoli and sometimes in the nuclei. Scale bar, 20 µm. Examples of stained tumors and controls are shown. e , The DHL cell line SUDHL6 was separated on denaturing acrylamide gels and analyzed by northern blotting. CD77 + tonsillar B cells were used as controls. As additional controls, and to unambiguously map the various 5′ end extended forms of 5.8S, HeLa cells depleted of RNA exosome subunits (EXOSC2 and EXOSC10) or of a cofactor (SKIV2L2) are used (left). HeLa cells depleted of (− lane), or overexpressing (+ lane), ZCCHC7 were also included. Results represent two or more independently performed biological replicates. f , Total RNA extracted from HeLa cells, SUDHL10 cells overexpressing ZCCHC7 (SUDHL10-OE), parental SUDHL10 (WT), PAX5 -TSS2 mut (SUDHL10 cells with PAX5 promoter DHL mutation) and two sets of PAX5 -TSS2 mut transfected with Exosc10 were processed as in e . Equal loading is shown with the U1 RNA control (bottom). Results represent two independently performed experiments. RPM, reads per million.

Article Snippet: SUDHL10 cells (CRL-2963) were purchased from the ATCC, cultured in the laboratory and modified through HDR to form the SUDHL10 PAX5 -TSS m ut cell line, as described elsewhere in this paper.

Techniques: RNA Expression, Mutagenesis, Staining, Immunohistochemistry, Northern Blot, Transfection, Control

Journal: Nature Genetics

Article Title: Noncoding mutations cause super-enhancer retargeting resulting in protein synthesis dysregulation during B cell lymphoma progression

doi: 10.1038/s41588-023-01561-1

Figure Lengend Snippet: (a) MYC-ZCCHC7 translocation identified in the DHL from P11. P11 represents a de novo DHL (no prior follicular lymphoma) selected for sequencing due to a high degree of ZCCHC7 expression observed on IHC, but not included in the main cohort of paired longitudinal samples. The translocation junction is observed between MYC downstream and ZCCHC7 downstream. (b) PVT1-ZCCHC7 translocation and CD274-ZCCHC7 rearrangement identified in SUDHL6 and RCK8 cell lines, respectively. The breakpoints in SUDHL6 cells were observed within the two genes, while the RING Zn-finger domain (light green) of ZCCHC7 remained in the fused transcript. Break points of the CD274-ZCCHC7 fusion in the RCK8 cell line are located in the 3′ UTR of CD274 and 32 kb downstream of ZCCHC7 . The fusion-supporting junction reads display ZCCHC7 in an antisense direction to CD274 . (c) Three ZCCHC7 translocations detected in RNAseq data from primary DLBCL tumors reported by Morin et al. . The PVT1-ZCCHC7 translocations were observed in two DHL patients. The majority of the functional domain (teal) of ZCCHC7 is preserved in the two fusion transcripts. The ZCCHC7- IGL translocation, which joined downstream of ZCCHC7 and IGLL5 exon 3, was detected in a DLBCL. All the RNAseq data supported gene fusions were detected by Arriba 2.3.0 and checked in IGV. (d) Investigation of the occurrence of the PAX5-ZCCHC7 gene fusion among n = 1,173 hematopoietic malignancies (HM) samples reported in the St. Jude Cloud ProteinPaint portal. The PAX5-ZCCHC7 gene fusions that connect intron 6 of PAX5 and intron 2 of ZCCHC7 in an antisense direction were reported in 17 cases of B-ALL. (e) Comparison of ZCCHC7 expression among four types of HM. B-ALL and B-ALL with known PAX5-ZCCHC7 fusions show the highest expression levels of ZCCHC7 compared to other types. The P -values were calculated using two-sided Wilcoxon rank-sum tests. The boxplots display the 25 th and 75 th percentiles and the median of each group of data. The whiskers represent the highest and lowest values within 1.5 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\times$$\end{document} × the inter-quartile range.

Article Snippet: SUDHL10 cells (CRL-2963) were purchased from the ATCC, cultured in the laboratory and modified through HDR to form the SUDHL10 PAX5 -TSS m ut cell line, as described elsewhere in this paper.

Techniques: Translocation Assay, Sequencing, Expressing, Functional Assay, Comparison

Journal: Nature Genetics

Article Title: Noncoding mutations cause super-enhancer retargeting resulting in protein synthesis dysregulation during B cell lymphoma progression

doi: 10.1038/s41588-023-01561-1

Figure Lengend Snippet: (a-c) Total RNA extracted from the indicated cells was separated by denaturing agarose gel electrophoresis, the gel was stained with ethidium bromide (panel a ), transferred to a nylon membrane and probed with radioactively-labeled anti-sense oligonucleotides specific to ITS1 (b) or 5′ ETS (c) SCR#1 is a non-targeting scrambled siRNA used to control HeLa depleted of EXOSC2, EXOSC10, and SKIV2L2. SCR#2 is non-targeting scrambled siRNA used to control HeLa depleted of ZCCHC7. The experiments represent results from 3 independently performed experiments. (d) Overexpression of Exosc10 ( Exosc10 -OE) in PAX5 -TSS2 mut SUDHL10 cells ( PAX5 -TSS2 mut ) rescues 5.8S + 40 rRNA processing. PAX5 -TSS2 mut SUDHL10 cells were transfected with a control vector that does not contain Exosc10 or with an Exosc10 expression vector also containing green fluorescent protein (GFP) (See western blot showing Exosc10 expression) and cells were sorted by red fluorescent protein (RFP) expression. The flow cytometric experiment was repeated three times. Total RNA was isolated from GFP positive cells and separated on a 7% acrylamide gel, transferred to a membrane and blotted with a probe against 5.8S+40 rRNA. The level of 5.8S+40 rRNA decreases following Exosc10 overexpression (lane 3), as compared to untransfected PAX5- TSS2 mut cells (lane 1), PAX5 -TSS2 mut cells containing the control vector (lane 2), or unmutated and untransfected SUDHL10 cells (lane 4).

Article Snippet: SUDHL10 cells (CRL-2963) were purchased from the ATCC, cultured in the laboratory and modified through HDR to form the SUDHL10 PAX5 -TSS m ut cell line, as described elsewhere in this paper.

Techniques: Agarose Gel Electrophoresis, Staining, Membrane, Labeling, Control, Over Expression, Transfection, Plasmid Preparation, Expressing, Western Blot, Isolation, Acrylamide Gel Assay

Journal: Nature Genetics

Article Title: Noncoding mutations cause super-enhancer retargeting resulting in protein synthesis dysregulation during B cell lymphoma progression

doi: 10.1038/s41588-023-01561-1

Figure Lengend Snippet: a , Schematic of nascent polypeptide synthesis analyses using OPP-ID (created with BioRender.com ). b , c , Volcano plots showing differences in abundance of OPP+ proteins, in SUDHL10 cells with ZCCHC7 -OE ( b ) or PAX5 -TSS2 mut ( c ), compared to the SUDHL10 WT cells. The proteins that are significantly upregulated or downregulated in ZCCHC7 -OE or PAX5 -TSS2 mut are highlighted in red or blue, respectively. d , Principal component analysis plot of the proteomics data from SUDHL10 WT cells (no inherent mutation or alterations in ZCCHC7 or PAX5 -TSS2), ZCCHC7 -OE and PAX5 -TSS2 m ut , with six biological replicates per group. Three replicates each were cultured with or without OPP (OPP+ and OPP−, respectively). Protein signals detected in the OPP+ group are shown as the union of OPP-labeled nascent proteins and nonspecifically captured background proteins. The OPP− data, representing protein signals from nonspecifically captured background proteins, were used as control to distinguish nascent proteins from background proteins. e , Two-way ANOVA test to evaluate the effect of genetic alteration ( ZCCHC7 -OE or PAX5 -TSS2 m ut ) and OPP label on proteomic intensity. The plot shows FDR adjusted P values of the two factors ( x axis for genetic alteration and y axis for OPP label) in the two-way ANOVA test. Each dot represents a protein and shows the abundance of corresponding proteins that decreased (left side) or increased (right side) in the ZCCHC7 -OE and PAX5 -TSS2 m ut cells. The upregulated/downregulated proteins were chosen by setting the adjusted genetic alteration P value < 0.01. Oncoproteins that increased and tumor suppressor proteins (TSPs) that decreased in SUDHL10 ZCCHC7 -OE and SUDHL10 PAX5 -TSS2 m ut cells are highlighted in red and blue, respectively. Two-sided Fisher’s exact tests were conducted to evaluate the relationship between oncoproteins/TSPs and upregulation/downregulation. The results show significant association of TSPs and downregulation with P = 0.0016. f , g , Polysome analyses of SUDHL10 cells and SUDHL10 PAX5 -TSS2 m ut cells. The distribution of IKZF3 mRNA ( g ) to heavier (highly translated fractions) decreases following PAX5 -TSS2 mutation. Gross proteome-wide translational perturbation is not observed in PAX5 -TSS2 m ut cells based on comparable polysome profiles ( f ), but specific mRNAs (for example, IKZF3 ) are translated differently ( g ). The data represent conclusions drawn from two independent biological replicates. ANOVA, analysis of variance. FDR, false discovery rate.

Article Snippet: SUDHL10 cells (CRL-2963) were purchased from the ATCC, cultured in the laboratory and modified through HDR to form the SUDHL10 PAX5 -TSS m ut cell line, as described elsewhere in this paper.

Techniques: Mutagenesis, Cell Culture, Labeling, Control

Journal: Nature Genetics

Article Title: Noncoding mutations cause super-enhancer retargeting resulting in protein synthesis dysregulation during B cell lymphoma progression

doi: 10.1038/s41588-023-01561-1

Figure Lengend Snippet: (a and b) Ranked gene set enrichment analysis (GSEA) of O-propargyl puromycin-mediated identification (OPP-ID) data using KEGG pathways collection. Genes (n = 5,176) corresponding to altered proteins were ranked in ascending order by two-way ANOVA test P -value of the genetic alteration factor on the x-axis in Fig. . Pathways that show FDR adjusted P -value < 0.05 are shown in ( a ). The sign of normalized enrichment score (NES) suggests downregulation (negative NES) or upregulation (positive NES) in the SUDHL10 ZCCHC7 -OE cells and SUDHL10 PAX5 TSS2 Mut cells compared to SUDHL10 WT cells. GSEA shows downregulation of proteins in ribosome, DNA replication, mismatch repair, and non-homologous end joining pathways ( b ). (c) Fold changes of nascent proteins in ZCCHC7 -OE or PAX5 TSS2 Mut SUDHL10 cells compared to the wild type SUDHL10 cells. Each dot represents a protein with significantly higher abundance in OPP+ than OPP- in a two-way ANOVA (Fig. ). 1,690 proteins in total were captured via OPP and evaluated for nascent protein level. P -value and rho of Spearman’s correlation test between the x-axis and y-axis show a significant positive correlation between fold change of proteins in ZCCHC7 -OE and PAX5 TSS2 Mut . Tumor suppressor proteins including IKZF3, CHEK1, etc show decreasing translation in both ZCCHC7 -OE cells and PAX5 TSS2 Mut cells. (d) Independent whole cell proteomics for triplicate cultures of CL-01 (non-neoplastic B cells), SUDHL6 (which overexpress ZCCHC7 due to chromosomal translocation), SUDHL10 cells (no inherent alterations in PAX5 -TSS2 or ZCCHC7 ) and ZCCHC7 -OE (SUDHL10 cells overexpressing ZCCHC7 ). Of the 3428 proteins with different protein expression between SUDHL6 and CL-01, 215 showed alterations also observed in ZCCHC7 -OE compared to SUDHL10. Some of these proteins are tumor suppressor proteins in B-NHL (for example IKZF3, TRAF3, etc.). (e and f) Polysome profiling of SUDHL10 WT and SUDHL10 ZCCHC7-OE ( e ) and analyses of IKZF3 mRNA distribution on the polysome ( f ). (g) ZCCHC7 transgene was introduced into SUDHL10, and stable lines isolated. ZCCHC7 protein expression is compared in ZCCHC7-OE and the SUDHL10 parental cell line. Three biological replicates were performed. (h) Evaluation of nascent levels of 20 proteins targeted by available therapeutics. The expression heatmap of the proteins in SUDHL10 and SUDHL10 PAX5 -TSS2 mut lymphoma cells is shown.

Article Snippet: SUDHL10 cells (CRL-2963) were purchased from the ATCC, cultured in the laboratory and modified through HDR to form the SUDHL10 PAX5 -TSS m ut cell line, as described elsewhere in this paper.

Techniques: Non-Homologous End Joining, Translocation Assay, Expressing, Isolation

Journal: Nature Genetics

Article Title: Noncoding mutations cause super-enhancer retargeting resulting in protein synthesis dysregulation during B cell lymphoma progression

doi: 10.1038/s41588-023-01561-1

Figure Lengend Snippet: A schematic representation of the contribution of aSHM to transformation from FL to DHL, along with various mechanisms of PAX5 / ZCCHC7 locus alterations that lead to aberrant rRNA processing and rewiring of lymphomagenic protein synthesis (created with BioRender.com ).

Article Snippet: SUDHL10 cells (CRL-2963) were purchased from the ATCC, cultured in the laboratory and modified through HDR to form the SUDHL10 PAX5 -TSS m ut cell line, as described elsewhere in this paper.

Techniques: Transformation Assay