Journal: RNA biology

Article Title: A conserved HOTAIRM1-HOXA1 regulatory axis contributes early to neuronal differentiation.

doi: 10.1080/15476286.2023.2258028

Figure Lengend Snippet: Figure 2. Depleting HOTAIRM1 hampers cell proliferation during neuronal differentiation. (A) schematic of the method used to differentiate cells. (B) Outline of the live cell imaging procedure used to track cell confluency. (C) NT2-D1 confluency changes in the absence (−RA) or presence (+RA) of retinoic acid as captured by live cell imaging. Timepoints reflect when pictures were taken as indicated in (B). (D) Representative phase contrast microscopy images of -RA or +RA NT2-D1 cells at the start (day 0) and end (day 3) of the differentiation time-course monitored in (C). (E) Steady-state levels of HOTAIRM1 lncRNA variants of collected samples monitored in (C). (F) Diagram indicating HOTAIRM1 regions targeted by siRNAs or quantified by RT-qPCR. 5’ to 3’ directionality is indicated by 7-methylguanosine cap (m7G) and poly(A) tail (pA); ‘E’ and ‘I’ represent exons and introns, respectively. (G) Outline of the live cell imaging procedure (top) used to track cell confluency (bottom). (H) Steady-state levels of HOTAIRM1 variants and HOXA1 in RNAi knockdown samples monitored in (I) and collected after 3 days of RA treatment. HOXA1 expression values were divided by 10 to display on the same scale as HOTAIRM1. (I) Confluency changes of NT2-D1 cells transfected with siRNAs targeting HOTAIRM1 or a negative control. Timepoints reflect when pictures were taken post RA treatment as indicated in (G). Yellow shading highlights exponential growth delay when HOTAIRM1 is depleted. Error bars in cell confluency graphs (C, I) show the standard error of the mean (sem) across 12 culture dish regions, whereas error bars in expression level graphs (E, H) are the standard deviations (stdevs) between at least 3 RT-qPCR measurements. Numbers above histogram bars are fold differences relative to corresponding siNC levels.

Article Snippet: Insertion of a 3XFlag-myc sequence into the endogenous HOXA1 gene by CRISPR/Cas9 editing HOXA1Δ guide oligos (top: 5’ - CACCGCTGACTACCTCCCA CTGAGG − 3’; bottom: 5’ - AAACCCTCAGTGGGAG GTAGTCAGC − 3’) were annealed and cloned into lentiCRISPRv2, a gift from Feng Zhang (Addgene plasmid #52961; http://n2t.net/addgene:52961; RRID: Addgene_52961) as per their recommendations [17].

Techniques: Live Cell Imaging, Microscopy, Quantitative RT-PCR, Knockdown, Expressing, Transfection, Negative Control

Journal: RNA biology

Article Title: A conserved HOTAIRM1-HOXA1 regulatory axis contributes early to neuronal differentiation.

doi: 10.1080/15476286.2023.2258028

Figure Lengend Snippet: Figure 3. HOTAIRM1 is required for proper neuronal differentiation. (A) SOX3 and HOTAIRM1 expression during a differentiation time-course in NT2-D1. Error bars are stdevs from three biological replicates, each with at least three measurements. *SOX3 values are relative to PGK1 whereas those for HOTAIRM1 are relative to actin (10−4). (B,C) SOX3 (B) or HOTAIRM1 and HOXA1 (C) expression in RNAi knockdown samples of NT2-D1 treated 3 days with RA. Expression values are from at least three measurements each from three independent biological replicates. Error bars represent the (sem). Numbers above histogram bars indicate fold differences relative to corresponding siNC samples. (D) SOX3 and HOTAIRM1 expression during an NCCIT differentiation time-course. Expression levels were measured as in (A). A biological replicate extending to Day 14 post-RA is in Supplemental Figure S2A. (E,F) SOX3 (E) or HOTAIRM1 and HOXA1 (F) expression in NCCIT RNAi knockdown samples treated 3 days with RA. Expression values are averages from at least three measurements and error bars are stdevs. Numbers above histogram bars are fold differences relative to corresponding siNC samples. A replicate of (E,F) collected 5 days post-RA is in Supplemental Figure S2B,C. A third biological replicate of Day 3 post-RA is given in Supplemental Figure S3E,F. Note that HOXA1 values in (C,F) were divided by 10 to be on a scale comparable to HOTAIRM1. (G) Expression of HOTAIRM1 and differentiation markers before and after RA induction for 3 days in NT2-D1. Error bars are stdevs from at least three measurements. HOTAIRM1 values were divided by 100 for direct comparison with other genes. (H) Expression in RNAi knockdown samples of NT2-D1 treated 3 days with RA. Expression values are from at least three measurements and error bars represent the stdev. HOTAIRM1 values were divided by 100 for direct comparison with other genes. Numbers above histogram bars indicate fold differences relative to corresponding siNC samples. RNA levels were quantified by RT-qPCR using primers listed in Supplemental Table S1. (I) Western blot analysis of SOX3 and HES1 protein levels in control (siNC) and HOTAIRM1-depleted (siM1.0, siM1.3) NCCIT cells treated with RA for 3 days.

Article Snippet: Insertion of a 3XFlag-myc sequence into the endogenous HOXA1 gene by CRISPR/Cas9 editing HOXA1Δ guide oligos (top: 5’ - CACCGCTGACTACCTCCCA CTGAGG − 3’; bottom: 5’ - AAACCCTCAGTGGGAG GTAGTCAGC − 3’) were annealed and cloned into lentiCRISPRv2, a gift from Feng Zhang (Addgene plasmid #52961; http://n2t.net/addgene:52961; RRID: Addgene_52961) as per their recommendations [17].

Techniques: Expressing, Knockdown, Comparison, Quantitative RT-PCR, Western Blot, Control

Journal: RNA biology

Article Title: A conserved HOTAIRM1-HOXA1 regulatory axis contributes early to neuronal differentiation.

doi: 10.1080/15476286.2023.2258028

Figure Lengend Snippet: Figure 4. HOTAIRM1 associates with the HOXA1 transcription factor in different cell types and species. (A) Diagram of the highly conserved, shared promoter region from which HOXA1 and HOTAIRM1 are transcribed on opposite strands. Base-wise conservation across 100 vertebrates by phyloP and base change locations between human and mouse is shown below. (B) HOXA1 and HOTAIRM1 RA induction kinetics in two neuronal differentiation models. RNA levels measured during RA (10 µM) induction time-courses in NT2-D1 (left) or NCCIT (right) using RT-qPCR. (C) Outline of the RNA immunoprecipitation (RIP) procedure. Note that RA is added 6 hours post-transfection, and that cells are collected either 2 or 3 days after RA treatment as specified in each panel. (D) Transfected Flag-hHOXA1 is specifically immunoprecipitated in RA-induced NCCIT cells. Western blot analysis of the input (1%) and RIP (5%) samples from one of the biological replicates analysed in (E), with eukaryotic translation elongation factor 2 (eEF2) probed as a control for specificity. (E) HOXA1 preferentially binds spliced HOTAIRM1 in RA-induced NCCIT cells. RIPs are from three biological replicates quantified by RT-qPCR. (F) Induction of mouse Hoxa1 and Hotairm1 by RA (1 µM) in P19 cells. (G) Transfected Flag- mHoxa1 is specifically immunoprecipitated in RA-induced P19 cells. Western blot analysis of the input (1%) and RIP (5%) samples corresponding to the assay shown in (H). (H) Mouse Hoxa1 preferentially binds mHotairm1 in RA-induced P19 cells. RT-qPCR measurements are from at least three PCRs with errors bars representing stdevs.

Article Snippet: Insertion of a 3XFlag-myc sequence into the endogenous HOXA1 gene by CRISPR/Cas9 editing HOXA1Δ guide oligos (top: 5’ - CACCGCTGACTACCTCCCA CTGAGG − 3’; bottom: 5’ - AAACCCTCAGTGGGAG GTAGTCAGC − 3’) were annealed and cloned into lentiCRISPRv2, a gift from Feng Zhang (Addgene plasmid #52961; http://n2t.net/addgene:52961; RRID: Addgene_52961) as per their recommendations [17].

Techniques: Quantitative RT-PCR, RNA Immunoprecipitation, Transfection, Immunoprecipitation, Western Blot, Control

Journal: RNA biology

Article Title: A conserved HOTAIRM1-HOXA1 regulatory axis contributes early to neuronal differentiation.

doi: 10.1080/15476286.2023.2258028

Figure Lengend Snippet: Figure 5. Depleting HOXA1 during differentiation delays NANOG downregulation. (A) NANOG and HOXA1 engage in positive auto- and negative cross-regulatory feedback mechanisms to control their gene expression. HOTAIRM1 (dashed) is proposed to work with HOXA1 as an RNP on the NANOG and HOXA1/HOTAIRM1 loci in both undifferentiated and RA-induced cell states; RARE = Retinoic Acid Response Element, ARE = Autonomous Response Element, NBS = NANOG Binding Site, HBS = HOXA1 Binding Site. (B) Steady-state NANOG and HOXA1 mRNA levels throughout RA-induced differentiation of NT2-D1 (left) or NCCIT (right) cells. *Relative HOXA1 mRNA levels were divided by 10 to display on the same scale with NANOG. (C,D) HOXA1 knockdown delays NANOG downregulation, blunts SOX3 upregulation, and impedes HOTAIRM1 induction during NT2-D1 differentiation. HOXA1 was RNAi-depleted and cells were collected 3 days after RA treatment (C) or every 24 hours along a time-course (D). A similar differentiation time-course for control (siNC) and HOXA1 (siA1) knockdown cells in NCCIT is shown in (E). RNA levels were measured by RT-qPCR from at least three measurements, with error bars showing stdevs. Numbers above histogram bars are fold differences relative to levels in siNC samples.

Article Snippet: Insertion of a 3XFlag-myc sequence into the endogenous HOXA1 gene by CRISPR/Cas9 editing HOXA1Δ guide oligos (top: 5’ - CACCGCTGACTACCTCCCA CTGAGG − 3’; bottom: 5’ - AAACCCTCAGTGGGAG GTAGTCAGC − 3’) were annealed and cloned into lentiCRISPRv2, a gift from Feng Zhang (Addgene plasmid #52961; http://n2t.net/addgene:52961; RRID: Addgene_52961) as per their recommendations [17].

Techniques: Control, Gene Expression, Binding Assay, Knockdown, Quantitative RT-PCR

Journal: RNA biology

Article Title: A conserved HOTAIRM1-HOXA1 regulatory axis contributes early to neuronal differentiation.

doi: 10.1080/15476286.2023.2258028

Figure Lengend Snippet: Figure 7. SOX2 repression is more sensitive than POU5F1 to HOTAIRM1 or HOXA1 depletion in early neuronal differentiation. (A) Steady-state SOX2 mRNA levels throughout RA-induced differentiation of NT2-D1 (left) or NCCIT (right) cells. (B) Depleting HOTAIRM1 during NT2-D1 (left) or NCCIT (right) differentiation curbs SOX2 downregulation. (C) Same as in B, except upon RNAi-mediated HOXA1 depletion. (D) Same as in (A), except that POU5F1 expression was measured. (E) HOTAIRM1 depletion does not significantly affect POU5F1 expression during either NT2-D1 (left) or NCCIT (right) differentiation. (F) HOXA1 depletion modestly affects POU5F1 expression during NT2-D1 (left) and NCCIT (right) differentiation. Knockdown levels of HOTAIRM1 and HOXA1 expression in (B) and (E) samples are shown in Figure 6B,C. HOXA1 knockdown levels and HOTAIRM1 expression in (C) and (F) samples are found in Figure 5D,E. All mRNA levels were measured by RT-qPCR and are from at least three measurements, with error bars representing stdevs. (G) Western blot analysis of HOXA1 and NANOG protein levels in either control (siNC), HOTAIRM1 (siM1) and/or HOXA1 (siA1) RNAi knockdown samples from a biological replicate RA differentiation time-course in NCCIT cells. The SOX2 and OCT4 protein levels and corresponding RNA levels measured by RT-qPCR and are presented in Supplemental Figure S6E,F.

Article Snippet: Insertion of a 3XFlag-myc sequence into the endogenous HOXA1 gene by CRISPR/Cas9 editing HOXA1Δ guide oligos (top: 5’ - CACCGCTGACTACCTCCCA CTGAGG − 3’; bottom: 5’ - AAACCCTCAGTGGGAG GTAGTCAGC − 3’) were annealed and cloned into lentiCRISPRv2, a gift from Feng Zhang (Addgene plasmid #52961; http://n2t.net/addgene:52961; RRID: Addgene_52961) as per their recommendations [17].

Techniques: Expressing, Knockdown, Quantitative RT-PCR, Western Blot, Control

Journal: RNA biology

Article Title: A conserved HOTAIRM1-HOXA1 regulatory axis contributes early to neuronal differentiation.

doi: 10.1080/15476286.2023.2258028

Figure Lengend Snippet: Figure 8. HOXA1 binding at HOXA, NANOG, and SOX2 gene loci is conserved in human cells. (A) summary schematic of the interaction network implicated with HOTAIRM1 and HOXA1. Dashed lines indicate functional connections not yet shown to occur directly. (B) Binding peaks of Flag-mHoxa1 in KH2 mES cells at 24 hours

Article Snippet: Insertion of a 3XFlag-myc sequence into the endogenous HOXA1 gene by CRISPR/Cas9 editing HOXA1Δ guide oligos (top: 5’ - CACCGCTGACTACCTCCCA CTGAGG − 3’; bottom: 5’ - AAACCCTCAGTGGGAG GTAGTCAGC − 3’) were annealed and cloned into lentiCRISPRv2, a gift from Feng Zhang (Addgene plasmid #52961; http://n2t.net/addgene:52961; RRID: Addgene_52961) as per their recommendations [17].

Techniques: Binding Assay, Functional Assay