Journal: Cancers

Article Title: A Combinatorial Regulatory Platform Determines Expression of RNA Polymerase III Subunit RPC7α ( POLR3G ) in Cancer

doi: 10.3390/cancers15204995

Figure Lengend Snippet: Primer sequences for molecular cloning.

Article Snippet: Real-time Quantitative PCR was performed with a TaqManTM Fast Advanced Master Mix (Catalog#4444557, Applied Biosystems, Waltham, Massachusetts, USA) and predesigned TaqMan Gene Expression Assays (20×; Catalog# 4331182, ThermoFisher, Waltham, MA, USA) for selected genes: Hs02786624_g1 (GAPDH); Hs99999903_m1 (ACTB); Hs04978644_g1 (POLR3G); Hs01113209_g1 (POLR3GL); Hs00153408_m1 (MYC); Hs00811069_g1 (MAX); Hs00940446_m1 (RARA); Hs01067640_m1 (RXRA); Hs01045973_m (ZNF207); Hs00399572_m1 (ZNF131); Hs00355782_m1 (CDKN1A); Hs00153277_m1 (CDKN1B); Hs00364847_m1 (CDK4); and Hs00153380_m1 (CCND2). mRNA abundances presented were determined as the relative fold change normalized against the geometric mean of two reference genes: GAPDH + ACTB.

Techniques: Cloning, Sequencing

Journal: Cancers

Article Title: A Combinatorial Regulatory Platform Determines Expression of RNA Polymerase III Subunit RPC7α ( POLR3G ) in Cancer

doi: 10.3390/cancers15204995

Figure Lengend Snippet: siRNA sequences.

Article Snippet: Real-time Quantitative PCR was performed with a TaqManTM Fast Advanced Master Mix (Catalog#4444557, Applied Biosystems, Waltham, Massachusetts, USA) and predesigned TaqMan Gene Expression Assays (20×; Catalog# 4331182, ThermoFisher, Waltham, MA, USA) for selected genes: Hs02786624_g1 (GAPDH); Hs99999903_m1 (ACTB); Hs04978644_g1 (POLR3G); Hs01113209_g1 (POLR3GL); Hs00153408_m1 (MYC); Hs00811069_g1 (MAX); Hs00940446_m1 (RARA); Hs01067640_m1 (RXRA); Hs01045973_m (ZNF207); Hs00399572_m1 (ZNF131); Hs00355782_m1 (CDKN1A); Hs00153277_m1 (CDKN1B); Hs00364847_m1 (CDK4); and Hs00153380_m1 (CCND2). mRNA abundances presented were determined as the relative fold change normalized against the geometric mean of two reference genes: GAPDH + ACTB.

Techniques: Sequencing, Concentration Assay

Journal: Cancers

Article Title: A Combinatorial Regulatory Platform Determines Expression of RNA Polymerase III Subunit RPC7α ( POLR3G ) in Cancer

doi: 10.3390/cancers15204995

Figure Lengend Snippet: An integrated survey of mRNA and chromatin correlates identifies candidate regulatory factors and sequence elements. ( a ) Cryo-EM structure highlighting major elements of the Pol III complex, including the stalk module (gold), RPC4-5 subcomplex (red), and RPC3-RPC6-RPC7 subcomplex (blue). Structure 7D59 re-colored in Mol*. ( b ) RPC7 subunits (blue/green) are highlighted for generalization; true structure corresponds to subunit RPC7α. Immunoblot analysis of commercial antibody specificity for RPC7α ( POLR3G ) and RPC7β ( POLR3GL ) in HEK293T cell lysates with ectopic POLR3G or POLR3GL overexpression. (−) non-transfected HEK293T cells. ( c ) Clinical outcome signatures for POLR3G and POLR3GL expression stratified by cancer subtype ( x -axis) and sorted by POLR3G z-score ( y -axis) association with favorable or unfavorable outcomes. Data acquired from tcga-survival.com version 2.0 . ( d ) Approach: co-expression correlation survey determines subtype and pan-cancer correlation scores between POLR3G and all other genes, with the goal of identifying candidate regulators broadly involved in POLR3G activity. ( e ) Approach: chromatin survey identifies 100 bp bins (±500 Kb) with significant correlation scores between chromatin accessibility and POLR3G expression in cancer, towards identifying candidate regulatory sequences related to POLR3G upregulation. ( f ) Co-expression correlation results from d: correlations are ranked ( x -axis) and z-transformed ( y -axis). Pluripotency factors (OCT4, NANOG, SOX2, and KLF4) and MAX are highlighted in gray. Significant positive (red) and negative (blue) correlates include ZNF131, ZNF207, and RARA and RXRA. Table highlights top 10 positive and negative correlates after subsetting for chromatin/transcription factors. ( g ) Chromatin correlation results from e: the most significant positive (red) DNA elements are identified within POLR3G , whereas significant negative (blue) DNA correlates are identified distal-downstream. Bins are scored with −log 10 (adjusted correlation p -value); regions with negative correlation coefficients are inverted along the y -axis. The top enriched motifs within positive (red) and negative (blue) bins were identified with STREME .

Article Snippet: Real-time Quantitative PCR was performed with a TaqManTM Fast Advanced Master Mix (Catalog#4444557, Applied Biosystems, Waltham, Massachusetts, USA) and predesigned TaqMan Gene Expression Assays (20×; Catalog# 4331182, ThermoFisher, Waltham, MA, USA) for selected genes: Hs02786624_g1 (GAPDH); Hs99999903_m1 (ACTB); Hs04978644_g1 (POLR3G); Hs01113209_g1 (POLR3GL); Hs00153408_m1 (MYC); Hs00811069_g1 (MAX); Hs00940446_m1 (RARA); Hs01067640_m1 (RXRA); Hs01045973_m (ZNF207); Hs00399572_m1 (ZNF131); Hs00355782_m1 (CDKN1A); Hs00153277_m1 (CDKN1B); Hs00364847_m1 (CDK4); and Hs00153380_m1 (CCND2). mRNA abundances presented were determined as the relative fold change normalized against the geometric mean of two reference genes: GAPDH + ACTB.

Techniques: Sequencing, Cryo-EM Sample Prep, Western Blot, Over Expression, Transfection, Expressing, Activity Assay, Transformation Assay

Journal: Cancers

Article Title: A Combinatorial Regulatory Platform Determines Expression of RNA Polymerase III Subunit RPC7α ( POLR3G ) in Cancer

doi: 10.3390/cancers15204995

Figure Lengend Snippet: A multimodal regulatory platform, including a gene-internal element linked to multiple transcription factors, promotes POLR3G expression and RPC7α abundance. ( a ) ChIP-seq profiles for known and candidate positive regulators of POLR3G expression, including pluripotency factors (OCT4 and NANOG), MYC/MAX, ZNF131, and ZNF207, overlayed on the POLR3G gene promoter and DNA bins with significant correlation scores (top). Height and shading denote the significance (−log 10 (adj. pval)) of positive (red) and negative (blue) chromatin correlation scores at each 100 bp bin. Gene track includes the dominantly expressed POLR3G isoform (black); alternate POLR3G transcript annotations (light gray); and MBLAC2 (dark gray). + strand orientation (right arrow) and − strand orientation (left arrow) shown. * All ChIP-seq profiles shown are composite signals derived from ChIP-atlas, with the exception of ZNF131 (ZBTB35 ChIP data in HEK293 cells ). Additional signals shown for TFs (PKNOX1, PBX2, and PBX4), histone marks (H3K4me3, H3K4me1, and H3K27ac), and annotated super enhancers (SEdb2.0) . ( b ) Context-specific enrichment for ATAC peak overlap with DNA coordinates featuring significant positive correlation with POLR3G expression (padj < 0.0001). ( c ) Context-specific enrichment for H3K4me1 ( x -axis) and H3K27ac ( y -axis) peak overlap with DNA coordinates featuring significant positive correlation with POLR3G expression (padj < 0.0001). Contexts with both high H3K4me1 and H3K27ac are highlighted. ( d ) Enrichment for ChIP-seq peak (TFs and other factors) overlap with DNA coordinates featuring significant positive correlation with POLR3G expression (padj < 0.0001). ( e ) Relative mRNA abundance of MYC and POLR3G (RPC7α) after siRNA mediated MYC knockdown in HEK293T. ( f , g ) Immunoblots and quantification of MYC, POLR3G (RPC7α), and β-Tubulin protein levels following MYC knockdown. ( h ) Dose–response RT−qPCR analysis of POLR3G (RPC7α) mRNA level following exposure of THP−1 monocytes to 10058-F4 (F4). ( i ) Immunoblot detection of MYC, RPC7α ( POLR3G ), and β-Tubulin protein abundance following F4 exposure. ( j ) Relative protein abundance of RPC7α ( POLR3G ) after F4 treatment. ( k ) RT−qPCR analysis of ZNF131 and POLR3G mRNA levels following siRNA-mediated ZNF131 knockdown. ( l , m ) Immunoblots and quantification of ZNF131 and POLR3G (RPC7α) protein levels following siRNA-mediated ZNF131 knockdown. ( n ) RT−qPCR analysis of ZNF207 and POLR3G mRNA level following siRNA-mediated ZNF207 knockdown. ( o , p ) Immunoblots and quantification of ZNF207 and POLR3G (RPC7α) protein levels following siRNA-mediated ZNF131 knockdown. Biological replicates = 3; two-group comparison ( e , g ) analyzed with t -test; multi-group comparison ( h , j ) analyzed with ANOVA; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

Article Snippet: Real-time Quantitative PCR was performed with a TaqManTM Fast Advanced Master Mix (Catalog#4444557, Applied Biosystems, Waltham, Massachusetts, USA) and predesigned TaqMan Gene Expression Assays (20×; Catalog# 4331182, ThermoFisher, Waltham, MA, USA) for selected genes: Hs02786624_g1 (GAPDH); Hs99999903_m1 (ACTB); Hs04978644_g1 (POLR3G); Hs01113209_g1 (POLR3GL); Hs00153408_m1 (MYC); Hs00811069_g1 (MAX); Hs00940446_m1 (RARA); Hs01067640_m1 (RXRA); Hs01045973_m (ZNF207); Hs00399572_m1 (ZNF131); Hs00355782_m1 (CDKN1A); Hs00153277_m1 (CDKN1B); Hs00364847_m1 (CDK4); and Hs00153380_m1 (CCND2). mRNA abundances presented were determined as the relative fold change normalized against the geometric mean of two reference genes: GAPDH + ACTB.

Techniques: Expressing, ChIP-sequencing, Derivative Assay, Knockdown, Western Blot, Quantitative RT-PCR, Quantitative Proteomics, Comparison

Journal: The EMBO Journal

Article Title: Parthenolide disrupts mitosis by inhibiting ZNF207/BUGZ-promoted kinetochore-microtubule attachment

doi: 10.1038/s44318-025-00469-2

Figure Lengend Snippet: ( A ) Volcano plot showing quantitative mass-spectrometry results. Dashed horizontal line shows the p -value cut-off ( p < 0.05) and vertical dashed lines indicate the upregulated/downregulated (competed/non-competed by free PTL) proteins. The green transparent region groups all the proteins that satisfy the p -value cut-off and are upregulated (competed) with a SILAC ratio higher than 2. N (number of experiments): 3. Statistical analysis was performed using unpaired t-test. ( B ) Representative spinning disk confocal time-series of mitosis in HeLa parental cells and HeLa stably expressing GFP-BUGZ undergoing indicated treatments. Scale bar: 10 µm. ( C ) Quantification of chromosome congression status and mitotic duration in HeLa parental cells and HeLa stably expressing GFP-BUGZ undergoing indicated treatments. Median is plotted for mitotic duration. N , n ( N = number of cells, n = number of experiments) for congression phenotype: HeLa + DMSO (44, 3), HeLa + 15 µM PTL (41, 3), HeLa GFP-BUGZ + DMSO (85, 3), HeLa GFP-BUGZ + 15 µM PTL (30, 3), HeLa GFP-BUGZ + 30 µM PTL (56, 3), HeLa GFP-BUGZ + 45 µM PTL (58, 3); N , n ( N = number of cells, n = number of experiments) for mitotic duration: HeLa + DMSO (40, 3), HeLa + 15 µM PTL (36, 3), HeLa GFP-BUGZ + DMSO (85, 3), HeLa GFP-BUGZ + 15 µM PTL (30, 3), HeLa GFP-BUGZ + 30 µM PTL (56, 3), HeLa GFP-BUGZ + 45 µM PTL (58, 3). .

Article Snippet: The BUGZ gene (human, NCBI RefSeq: NM_001032293.3 ) encoding wild-type BUGZ and C54A mutant were commercially synthesized (GenScript) as siRNA-resistant sequences into a pGenDONR vector (pGenDONR-BUGZ and pGenDONR-BUGZ C54A).

Techniques: Mass Spectrometry, Multiplex sample analysis, Stable Transfection, Expressing

Journal: The EMBO Journal

Article Title: Parthenolide disrupts mitosis by inhibiting ZNF207/BUGZ-promoted kinetochore-microtubule attachment

doi: 10.1038/s44318-025-00469-2

Figure Lengend Snippet: ( A ) Representative spinning-disk confocal time-series of mitosis in HeLa cells stably expressing GFP-BUGZ and infected with adenovirus to express H2B-RFP. Scale bar: 10 µm. ( B ) Representative spinning-disk confocal time-series of mitosis in control, 15 µM PTL- and siBUGZ-treated U2OS cells stably expressing H2B-GFP/mScarlet-α-tubulin. Scale bar: 10 µm.

Article Snippet: The BUGZ gene (human, NCBI RefSeq: NM_001032293.3 ) encoding wild-type BUGZ and C54A mutant were commercially synthesized (GenScript) as siRNA-resistant sequences into a pGenDONR vector (pGenDONR-BUGZ and pGenDONR-BUGZ C54A).

Techniques: Stable Transfection, Expressing, Infection, Control

Journal: The EMBO Journal

Article Title: Parthenolide disrupts mitosis by inhibiting ZNF207/BUGZ-promoted kinetochore-microtubule attachment

doi: 10.1038/s44318-025-00469-2

Figure Lengend Snippet: ( A ) Fluorencence and Coomassie staining of immunoprecipitated FLAG-BUGZ from HEK 293T cells treated either with DMSO or 15 µM alkyne-PTL. ( B ) Representative spinning-disk confocal maximum projections of click-based imaging of 15 µM fluor-488-alkyne-PTL in nocodazole arrested U2OS cells co-stained with α-tubulin and CENP-C, as markers for microtubules and kinetochores, respectively. Scale bar: 10 µm. ( C ) Quantification of the relative levels of 15 µM fluor-488-alkyne-PTL at kinetochores. N , n (number of cells, number of experiments): siNT (38, 4) siBUGZ (40, 4). *** p ≤0.001. ( D ) Representative confocal maximum projections of BUB1 immunostainings in nocodazole arrested U2OS cells treated with DMSO or 15 µM PTL. Scale bar: 10 µm. ( E ) Quantification of the relative levels of BUB1 at kinetochores. N , n ( N = number of cells, n = number of experiments): DMSO (40, 4), 15 µM PTL (40, 4). ( F ) Extracted ion chromatograms for peptides with and without PTL modification at Cys54 (red, and green, respectively). ( G ) Extent of PTL-modification at Cys54 from 3 independent experiments. ( H ) AlphaFold 3 model of BUGZ Zinc Finger domains and Cys54 positioning showing the catalytic mechanism leading to PTL selectivity. Replicates are color-coded for all quantifications. Data in ( C ) and ( E ) are presented as mean ± SD values, while data in ( G ) are presented in mean ± SEM values. Statistical analysis was performed by unpaired t-test with Welch’s correction in ( C ) and Mann-Whitney test in ( E ). .

Article Snippet: The BUGZ gene (human, NCBI RefSeq: NM_001032293.3 ) encoding wild-type BUGZ and C54A mutant were commercially synthesized (GenScript) as siRNA-resistant sequences into a pGenDONR vector (pGenDONR-BUGZ and pGenDONR-BUGZ C54A).

Techniques: Staining, Immunoprecipitation, Imaging, Modification, MANN-WHITNEY

Journal: The EMBO Journal

Article Title: Parthenolide disrupts mitosis by inhibiting ZNF207/BUGZ-promoted kinetochore-microtubule attachment

doi: 10.1038/s44318-025-00469-2

Figure Lengend Snippet: ( A ) Fluorencence and Comassie staining of immunoprecipitated FLAG empty and FLAG-BUGZ from HEK 293T cells treated alkyne-PTL. ( B ) Representative confocal images of click-based imaging of 5 µM fluor-Cy5-alkyne-PTL in HeLa GFP-BUGZ cells under the indicated conditions. Scale bar: 10 µm. ( C ) Scatter plot showing the intensity of BUGZ (x-axis) and 5 µM fluor-Cy5-alkyne-PTL (y-axis) at individual kinetochores from the indicated conditions in ( B ). Each dot represents a single kinetochore. A Pearson correlation line is shown for the correlation between BUGZ and fluor-Cy5-alkyne-PTL levels. ( D ) Quantification of 5 µM fluor-Cy5-alkyne-PTL intensity at kinetochores normalized to CENP-C intensity for the conditions indicated in ( B ). N , n (number of cells, number of experiments): siNT (19, 3) siBUGZ (21, 3). **** p ≤ 0.0001. Replicates are color coded. Data are presented as mean ± SD values from three independent replicates. Statistical analysis was performed using unpaired t-test. ( E ) Immunoblot for BUGZ depletion efficiency in HeLa GFP-BUGZ cells. ( F ) Illustration of domain architecture of BUGZ. ( G ) Western-blot with anti-BUGZ antibody of in cellulo GFP-Trap pulldown sample from HeLa cells stably expressing GFP-BUGZ treated with 50 µM PTL. ( H ) Extracted ion chromatograms and MS-MS spectra for peptides with and without PTL modification at Cys54 (red and green, respectively) from the GFP-Trap sample shown in ( G ).

Article Snippet: The BUGZ gene (human, NCBI RefSeq: NM_001032293.3 ) encoding wild-type BUGZ and C54A mutant were commercially synthesized (GenScript) as siRNA-resistant sequences into a pGenDONR vector (pGenDONR-BUGZ and pGenDONR-BUGZ C54A).

Techniques: Staining, Immunoprecipitation, Imaging, Western Blot, Stable Transfection, Expressing, Tandem Mass Spectroscopy, Modification