Journal: Non-coding RNA Research

Article Title: A novel enhancer RNA, Hmrhl, positively regulates its host gene, phkb, in chronic myelogenous leukemia

doi: 10.1016/j.ncrna.2019.08.001

Figure Lengend Snippet: Expression and coding potential analysis of Hmrhl. a. Quantitative real time PCR analysis of Hmrhl expression showed that it is expressed in all human tissues (Brain, Heart, Kidney, lung, liver, pancreas, spleen, thymus, small intestine, colon, skeletal muscle, testes, prostate, ovary, placenta, leukocyte, from left to right) examined. Lowest expression was found in skeletal muscle (SM) which was taken as control, the level of which was considered as 1 and all others were plotted in comparison to it. Highest expression was seen in spleen (spln) followed by pancreas (Pnc), testis (Tst) and other tissues. b. Northern blot detection of Hmrhl. Total RNA from HEK 293T and K562 cell lines were separated on agarose gel and subsequently hybridized with DIG labelled Hmrhl specific riboprobe to detect the transcript (i). In parallel, methylene blue staining was used to determine the size of HMRHL, using 28 S rRNA (5 kb) and 18s rRNA (1.9 kb) as reference (ii). Note that the size of Hmrhl is similar to that of 28s rRNA, revealing that Hmrhl is about 5 kb in size. c. Protein-coding potential as determined by Broad Institute's PhyloCSF data and visualized in UCSC Genome Browser, showing that Hmrhl has no coding potential. d. Circular phylogenetic tree built in iTOL (Interactive Tree of Life).

Article Snippet: Since Hmrhl locus exhibited enhancer properties in K562 Chronic Myelogenous Leukemia cells, we examined the expression profile of Hmrhl across various human cancers using a cancer specific cDNA panel (Origene, USA) by real time qPCR.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Comparison, Northern Blot, Agarose Gel Electrophoresis, Staining

Journal: Non-coding RNA Research

Article Title: A novel enhancer RNA, Hmrhl, positively regulates its host gene, phkb, in chronic myelogenous leukemia

doi: 10.1016/j.ncrna.2019.08.001

Figure Lengend Snippet: Hmrhl locus exhibits hallmarks of enhancer. a. ENCODE data visualized through Integrated Genome Viewer (IGV) for DNase hypersensitive sites, p300 binding, enhancer specific histone marks, H3K27Ac and H3K4Me1 and the promoter specific histone mark, H3K4Me3 at the 5′ end of Hmrhl, only in K562 but not in GM12878 cells. Note the two prominent peaks (red) for the enhancer mark H3K27Ac in K562. b-c. Chromatin immunoprecipitation with Ab8895 (anti-H3K4Me1 antibody) and Ab4729 (anti-H3K27Ac antibody) followed by qPCR in K562 cells. Note the enrichment of both the enhancer marks at the 5′ end of Hmrhl in the IP fraction as compared to input/PIS/gene desert region (GD), that serves as a negative control.

Article Snippet: Since Hmrhl locus exhibited enhancer properties in K562 Chronic Myelogenous Leukemia cells, we examined the expression profile of Hmrhl across various human cancers using a cancer specific cDNA panel (Origene, USA) by real time qPCR.

Techniques: Binding Assay, Chromatin Immunoprecipitation, Negative Control

Journal: Non-coding RNA Research

Article Title: A novel enhancer RNA, Hmrhl, positively regulates its host gene, phkb, in chronic myelogenous leukemia

doi: 10.1016/j.ncrna.2019.08.001

Figure Lengend Snippet: Hmrhl locus exhibits hallmarks of enhancer contd. a. Encode data shows the binding of various transcription and PolII at the 5′ end of Hmrhl. We have retained the H3K27Ac peaks in this figure also for a reference. b. Schematic for chromatin interaction analysis (ChiaPET data) for Hmrhl. The large purple-black peak representing histone marks on the extreme left denotes the promoter of phkb gene while the small purple peak at the far right represents the 5'end of Hmrhl. ChiaPET data shows the interaction of Hmrhl locus with phkb promoter, as represented by two black boxes (blue arrows) connected by a black line in b. The Hmrhl locus is expanded below in c , showing that this locus has enhancer properties only in K562 cell line (orange-yellow color), but not in other cell lines like GM12878, HepG2 or hESC. Genomic segments are colour coded by ENCODE as denoted in d , with red colour signifying active promoter ( phkb promoter at far left, black arrow in b ) while orange colour represents active enhancer at Hmrhl locus at far right (red arrow in b ).

Article Snippet: Since Hmrhl locus exhibited enhancer properties in K562 Chronic Myelogenous Leukemia cells, we examined the expression profile of Hmrhl across various human cancers using a cancer specific cDNA panel (Origene, USA) by real time qPCR.

Techniques: Binding Assay

Journal: Non-coding RNA Research

Article Title: A novel enhancer RNA, Hmrhl, positively regulates its host gene, phkb, in chronic myelogenous leukemia

doi: 10.1016/j.ncrna.2019.08.001

Figure Lengend Snippet: Hmrhl is differentially expressed in various cancers. a. Expression of Hmrhl in various normal and cancer samples as observed by qPCR. Note that Hmrhl is highly upregulated in several lymphoma samples (bracket) in comparison to normal range (arrow). In fact, of all cancers, the highest levels of Hmrhl are seen in some of the lymphoma samples. b-c. qPCR analysis of Hmrhl and PHKB expression showing that both are over expressed in K562 leukemia condition as compared to GM12878 normal lymphocytes.

Article Snippet: Since Hmrhl locus exhibited enhancer properties in K562 Chronic Myelogenous Leukemia cells, we examined the expression profile of Hmrhl across various human cancers using a cancer specific cDNA panel (Origene, USA) by real time qPCR.

Techniques: Expressing, Comparison

Journal: Non-coding RNA Research

Article Title: A novel enhancer RNA, Hmrhl, positively regulates its host gene, phkb, in chronic myelogenous leukemia

doi: 10.1016/j.ncrna.2019.08.001

Figure Lengend Snippet: Hmrhl functions as enhancer RNA for phkb gene. a. Lucifaerase assay showing the intense signal of reporter activity in K562 cells with insert 3 cloned in enhancer vector. Note the low level of luciferase signal obtained with insert 2 both with promoter and enhancer vectors. b. siRNA (Sigma) mediated down-regulation of Hmrhl causes down-regulation of PHKB in K562 cells treated with Hmrhl specific siRNA pool as compared to control cells without transfection and cells treated with scrambled siRNA as negative control. c-d. Smart pool siRNA (Dharmacon) were used against the Hmrhl region to downregulate Hmrhl and subsequently expression level of PHKB gene were checked by qPCR in both K562 and GM12878 cell lines. Scrambled siRNA was used as a negative control. Note the down regulation of PHKB only in K562.

Article Snippet: Since Hmrhl locus exhibited enhancer properties in K562 Chronic Myelogenous Leukemia cells, we examined the expression profile of Hmrhl across various human cancers using a cancer specific cDNA panel (Origene, USA) by real time qPCR.

Techniques: Activity Assay, Clone Assay, Plasmid Preparation, Luciferase, Control, Transfection, Negative Control, Expressing

Journal: Cell Genomics

Article Title: CRISPR activation screens map the genomic landscape of cancer glycome remodeling

doi: 10.1016/j.xgen.2026.101139

Figure Lengend Snippet: A CRISPR activation screening strategy for identifying genetic drivers of Siglec ligand expression (A) Diagram depicts the mechanism for inhibition of anticancer immunity by Siglec receptors. (B) The chemical structure of Siglec-7-binding O-linked glycans is depicted. (C) The strategy for overexpression of target genes by CRISPR activation (CRISPRa) is depicted. (D) Representative flow cytometry plots depict Siglec-Fc staining of K-562-CRISPRa cells. (E) Workflow of a FACS-based CRISPR screening strategy for identifying regulators of Siglec-Fc binding.

Article Snippet: Over 50% of these hits were natively expressed at extremely low levels in K-562 cells (Human Protein Atlas, mRNA transcripts per million [TPM] < 10) ( ).

Techniques: CRISPR, Activation Assay, Expressing, Inhibition, Binding Assay, Over Expression, Flow Cytometry, Staining

Journal: Cell Genomics

Article Title: CRISPR activation screens map the genomic landscape of cancer glycome remodeling

doi: 10.1016/j.xgen.2026.101139

Figure Lengend Snippet: Overexpression of specific cell surface glycoproteins increases the binding of Siglec-7 and Siglec-9 (A) Heatmap depicts CasTLE scores for top-ranked cell surface protein hits. (B) Heatmap depicts CasTLE scores for all genes in the MUC family of mucin-type O-glycoproteins. (C) Representative flow cytometry plot depicts gating strategy for K-562-CRISPRa cells transduced with a CD44 sgRNA and stained with an anti-CD44 antibody. NT sgRNA indicates cells transduced with a non-targeting sgRNA (sgNT). (D) Representative flow cytometry plot depicts staining of sgNT-transduced K-562 CRISPRa cells and CD44-overexpressing K-562 CRISPRa cells with Siglec-9-Fc. (E) The normalized median fluorescence intensity (MFI) of Siglec-9 staining in sgNT-transduced K-562-CRISPRa cells and CD44-overexpressing K-562 CRISPRa cells is shown. (F) Representative flow cytometry plot depicts staining of sgNT-transduced K-562 CRISPRa cells and CSPG4-overexpressing K-562 CRISPRa cells with Siglec-9-Fc. (G) The normalized MFI of Siglec-9 staining in sgNT-transduced K-562-CRISPRa cells and CSPG4-overexpressing K-562 CRISPRa cells is shown. (H) Graph depicts the number of N-linked and chondroitin sulfate glycosylation sites on Siglec-9 screen hits (UniProt). Statistical significance was determined using a two-tailed t test. ∗∗ p < 0.01 and ∗ p < 0.05. Mean values are plotted, and the error bars indicate the SEM.

Article Snippet: Over 50% of these hits were natively expressed at extremely low levels in K-562 cells (Human Protein Atlas, mRNA transcripts per million [TPM] < 10) ( ).

Techniques: Over Expression, Binding Assay, Flow Cytometry, Transduction, Staining, Fluorescence, Glycoproteomics, Two Tailed Test

Journal: Cell Genomics

Article Title: CRISPR activation screens map the genomic landscape of cancer glycome remodeling

doi: 10.1016/j.xgen.2026.101139

Figure Lengend Snippet: Siglec-10 displays broad specificity for N-linked sialoglycans (A and B) Representative flow cytometry plots depict staining of (A) K-562-CRISPRa cells and (B) THP-1 cells with an anti-CD24 antibody and Siglec-10-Fc. (C) The biosynthetic pathways for sialylation of N-linked glycans are shown. (D) Heatmap depicts the average mRNA expression of key glycosyltransferase hits in cell lines derived from B cell acute lymphoblastic leukemia (B-ALL), T cell acute lymphoblastic leukemia (T-ALL), acute myeloid leukemia (AML), and multiple myeloma. (E) Representative flow cytometry plots depict staining of OCI-AML-2 WT and ST3GAL4 KO cells with Siglec-10-Fc. (F) The normalized MFI of Siglec-10 staining in OCI-AML-2 WT and ST3GAL4 KO cells is shown. (G) Representative flow cytometry plot depicts co-staining of OCI-AML-2 MGAT1 KO cells with L-PHA and Siglec-10-Fc. (H) Graph indicates MFI of Siglec-9-Fc and Siglec-10-Fc staining in the WT (L-PHA+) and MGAT1 KO (L-PHA−) populations. (I) Representative flow cytometry plot depicts co-staining of OCI-AML-2 C1GALT1 KO cells with DBA and Siglec-10-Fc. (J) Graph indicates MFI of Siglec-9-Fc and Siglec-10-Fc staining in the WT (DBA−) and C1GALT1 KO (DBA+) populations. (K) Representative flow cytometry plot depicts staining of MM1S ST6GAL1 WT and KO cells with Siglec-10-Fc. (L) The normalized MFI of Siglec-10 staining in MM1S WT and ST6GAL1 KO cells is shown. (M) Representative flow cytometry plot depicts staining of sgNT-transduced K-562 CRISPRa cells and CD44-overexpressing K-562 CRISPRa cells with Siglec-10-Fc. (N) The normalized MFI of Siglec-10-Fc staining in sgNT-transduced K-562 CRISPRa cells and CD44-overexpressing K-562 CRISPRa cells is shown. (O) Representative flow cytometry plot depicts staining of sgNT-transduced K-562 CRISPRa cells and CSPG4-overexpressing K-562 CRISPRa cells with Siglec-10-Fc. (P) The normalized MFI of Siglec-10-Fc staining in sgNT-transduced K-562 CRISPRa cells and CSPG4-overexpressing K-562 CRISPRa cells is shown. Statistical significance was determined using a two-tailed t test. ∗∗ p < 0.01 and ∗ p < 0.05. NS, not significant. Mean values are plotted, and the error bars indicate the SEM.

Article Snippet: Over 50% of these hits were natively expressed at extremely low levels in K-562 cells (Human Protein Atlas, mRNA transcripts per million [TPM] < 10) ( ).

Techniques: Flow Cytometry, Staining, Expressing, Derivative Assay, Two Tailed Test