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Glia-specific enhancers and chromatin structure regulate NFIA expression and glioma tumorigenesis

Nature Neuroscience volume 20, pages 1520–1528 (2017)Cite this article

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Abstract

Long-range enhancer interactions critically regulate gene expression, yet little is known about how their coordinated activities contribute to CNS development or how this may, in turn, relate to disease states. By examining the regulation of the transcription factor NFIA in the developing spinal cord, we identified long-range enhancers that recapitulate NFIA expression across glial and neuronal lineages in vivo. Complementary genetic studies found that Sox9–Brn2 and Isl1–Lhx3 regulate enhancer activity and NFIA expression in glial and neuronal populations. Chromatin conformation analysis revealed that these enhancers and transcription factors form distinct architectures within these lineages in the spinal cord. In glioma models, the glia-specific architecture is present in tumors, and these enhancers are required for NFIA expression and contribute to glioma formation. By delineating three-dimensional mechanisms of gene expression regulation, our studies identify lineage-specific chromatin architectures and associated enhancers that regulate cell fate and tumorigenesis in the CNS.

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Figure 1: NFIA enhancers are regulated by lineage-specific mechanisms.
Figure 2: Brn2, Sox9 and Isl1 regulate NFIA expression in the spinal cord.
Figure 3: Enhancers demonstrate differential architecture at the NFIA locus.
Figure 4: Gliogenic chromatin loop forms before NFIA induction.
Figure 5: Gliogenic chromatin architecture is present in mouse models of glioma.
Figure 6: Glial enhancers are required for NFIA expression and tumorigenesis.

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Acknowledgements

This work was supported by grants from the National Institutes of Health (NS071153 to B.D. and K01CA190235 and 5-T32HL092332-08 to S.G.), Cancer Prevention Research Institute of Texas (RP150334 and RP160192 to B.D. and C.J.C.), and Sontag Foundation (B.D.).

Author information

Author notes

  1. Jeffrey C Carlson and Wenyi Zhu: These authors contributed equally to this work.

Authors and Affiliations

  1. Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, Texas, USA

    Stacey M Glasgow, Jeffrey C Carlson, Wenyi Zhu, Lesley S Chaboub, Peng Kang, Brittney E Lozzi & Benjamin Deneen

  2. Program in Developmental Biology, Baylor College of Medicine, Houston, Texas, USA

    Jeffrey C Carlson, Lesley S Chaboub & Benjamin Deneen

  3. Neurological Research Institute at Texas Children's Hospital, Houston, Texas, USA

    Hyun Kyoung Lee & Benjamin Deneen

  4. Department of Pediatrics, Pediatric Neuroscience Research Program, Papé Family Pediatric Research Institute, Portland, Oregon, USA

    Yoanne M Clovis & Soo-Kyung Lee

  5. Department of Cell and Developmental Biology, Oregon Health & Science University, Portland, Oregon, USA

    Yoanne M Clovis & Soo-Kyung Lee

  6. School of Medicine/HHMI, University of California San Diego, San Diego, California, USA

    Robert J McEvilly & Michael G Rosenfeld

  7. Division of Biostatistics, Dan L Duncan Cancer Center, Houston, Texas, USA

    Chad J Creighton

  8. Department of Pathology, Texas Children's Hospital, Houston, Texas, USA

    Carrie A Mohila

  9. Department of Neuroscience, Baylor College of Medicine, Houston, Texas, USA

    Benjamin Deneen

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Contributions

S.M.G., P.K., and B.D., conceived the project, designed the experiments. S.M.G. and B.D. wrote the manuscript. S.M.G., J.C.C., W.Z., P.K., H.K.L., L.S.C., and B.E.L. performed the experiments. B.D., S.M.G., W.Z., J.C.C., and C.A.M. analyzed the data. R.M., M.G.R., S.-K.L., and Y.M.C. provided essential reagents. C.J.C. provided bioinformatic analysis.

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Correspondence to Benjamin Deneen.

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Integrated supplementary information

Supplementary Figure 1 Comparison of mouse and chick NFIA enhancers across chick and mouse systems.

(A-B) Mouse and chick NFIA locus. The black lines represent highly conserved regions, ranging being 100-300bp, that are <70% conserved. The red boxes in B represent the 13 candidate enhancer elements that contain clusters of these conserved regions. (C) Mouse NFIA locus, depicting the locations of e96, e123, and e161 relative to the TSS and exon1. (D) Chick NFIA locus demonstrating locations of the enhancers and TSS/exon1. (E) Chick/Mouse alignment of conserved regions in each of the enhancer. Mouse enhancer regions are: E96:Chr4: 97665168-97670908; E123:Chr4:97717697-97721476; and E161:Chr4: 7806020-97806814.

Supplementary Figure 2 Deletion-mapping the e161 response region in the embryonic chick spinal cord.

(A-D) Chick spinal cord electroporated with e161 regions depicted in AA and harvested at E6; note fragment 2 is 1.7kb in length. (E-J) Chick spinal cords electroporated with regions (DC1-3) derived from within fragment 2 of e161 (see map in AA) and harvested at E6; note that DC1-2 are each 500bp in length. (K-R) Chick spinal cords electroporated with regions R1-R4 derived from within DC1 (see map in BB) and harvested at E6; note that R1-4 are each 125bp in length. (S-Z) Chick spinal cords electroporated with nested 30bp deletions of R3, labeled S1-S4 (see map in BB) and harvested at E6. (AA) Map of e161 and the relative locations of fragments 1 and 2 and DC1-3. (BB) Map of DC1 region of e161 and the relative locations of R1-4 and nested deletions within R3 (S1-4). Note that each enhancer construct was co-electroporated in trans with a CMV-Cherry to report efficient electroporation. Images are representative of at least four different chick embryos, 8 sections analyzed per embryo. Scale bars are 100um.

Supplementary Figure 3 Deletion-mapping of the e96 response region in the embryonic chick spinal cord.

(A-G) Chick spinal cord electroporated with e96 regions depicted in P; note that each fragment in 1kb in length. (H-O) Mutation analysis of the Isl1/Lhx3 sites located within fragment 3. (P) Map of e96 and the relative locations of the fragments and the Isl1/Lhx3 binding sites within fragment 3. (Q-Y) Ectopic overexpression of HA-Sox9 (Q-S), HA-Brn2 (T-V), and Isl1 (W-Y). Each of these images is representative of the ectopic over expression of the given transcription factor and are taken from sections that are adjacent to the images shown in Figure 1. Note that each enhancer construct was co-electroporated in trans with a CMV-Cherry to report efficient electroporation. Images are representative of at least four different chick embryos, 8 sections analyzed per embryo. All staining in figure is immunofluorescence. Scale bars are 100um.

Supplementary Figure 4 Analysis of Sox9 and Brn2 mutants; NFIA expression in MNs.

(A-F) Analysis of Sox9 and Brn2 expression in mutant embryos at E12.5; (A-B) analysis of the Sox9 mutant, (C-D) analysis of the Brn2 mutant, (E-F) analysis of the Sox9;Brn2 compound mutant. These stainings confirm deletion of the desired protein and that there is not a compensatory upregulation of the other protein. (G-J) Expression of NFIA in Isl1-expresing MN populations at E12.5. NFIA is co-expressed with Isl1/2 in the brachial level of the spinal cord in MMC, LMC(m), and LMC(l) motor columns. (K) Immunoprecipitation of E12.5 spinal cord with Brn2 antibodies, reinforces biochemical association between Sox9 and Brn2; please see figure 2D for Sox9-immunopreciptation. All staining in figure is immunofluorescence. Scale bars are 100um.

Supplementary Figure 5 Relative location of primers used for ChIP assays

White lines in e96, e123, and e161 depict the associated transcription factor response site (e96, Lhx3/Isl1; e123, Sox9; e161, Brn2). Arrows depict primers used for ChIP assays; note that primers encompass the response regions, thus ChIP assays in Figure 3 are targeting the associated, transcription factor response regions. (E) Sox9 chromatin immunoprecipitation from E12.5 spinal cord showing that Sox9 associates with e123/161 and not e96. *p=0.005 (F) Brn2 chromatin immunoprecipitation from E12.5 spinal cord showing that Brn2 associates with e123/161 and not e96. *p=0.0098 (G) Isl1 chromatin immunoprecipitation from E12.5 spinal cord showing that Isl1 associates with e96 and not e123/e161. *p=0.003

Supplementary Figure 6 Human glioma expression characteristics

(A) Correlation of expression levels between NFIA, Sox9, Brn2, Isl1, and Lhx3 in human glioma was assessed using Spearman's correlation, with the gene expression data derived from 403 cases of GBM (Brennan, et al. 2013). (B-C) NFIA and Sox9, Brn2 expression is strongly correlated in human glioma, both GBM (Blue Dots) and Low Grade Glioma (LGG) (Red Dots). Spearman’s correlation coefficient plots are derived from human glioma RNA-Seq expression profiles from TCGA datasets representing a total of 667 low- and high-grade glioma samples (Ceccarelli, et al. 2016). (D) Correlation of expression levels between Lhx3/NFIA, Isl1/NFIA, Sox9/NFIA and Brn2/NFIA in human glioma was assessed using Spearman's correlation, with the gene expression data derived from 667 GBM and LGG cases.

Supplementary Figure 7 In utero electroporation-mediated mouse models of glioma.

(A-B) CAG-GFP controls. (C-D) GFP-Ras images of whole brain and cross-section at P14. Arrows are electroporated cortex; note tumor infiltration in (D). (G) Schematic of in utero electroporation model and tumor timeline. (E-F) H&E staining of glioma demonstrate pathological hallmarks of malignant glioma. (H-I) GFP Images from CRISPR/IUE whole brain and tumors at P70 and associated H&E staining showing pathology resembling malignant glioma (J). Scale bars are 100um.

Supplementary Figure 8 Analysis of e123- and e161-edited mouse glioma

Analysis of H&E, Sox9, Brn2, and NFIA expression in PB-Ras glioma model under the following conditions: (A-D) px330-control, (F-I) CRISPR deletion of both e123/e161, (K-N) CRISPR deletion of e123, and (P-S) CRISPR deletion of e161. Note that NFIA expression is only decreased in the compound e123/e161 deletion and that Sox9/Brn2 expression is unaffected. While the rate of tumor growth is greatly attenuated in the e123/e161 compound deletion, the tumors still resemble malignant glioma (F). Each experimental condition was performed on three independent mouse litters, resulting in a total of 15-20 mice bearing tumors for each condition. Images are representative for each of the conditions. (T-W) Expression of NFIA, Sox9, and Brn2 in our CRISPR/IUE model. Samples were analyzed at P70. (X-CC) Analysis of glial developmental markers GLAST and FABP7 in the PB-Ras glioma model. Note that there are no gross differences in the expression of these glial developmental markers in the enhancer deletion tumors and controls (X, Z v. AA, CC). (DD-GG) Bioluminescence imaging of PB-RAS tumors at P14 from the px330-control and enhancer deletion experimental conditions. (HH) Quantification of bioluminescence imaging of P14 PB-Ras tumors containing CRISPR mediated deletions in e123 and e161. *p=0.005. A comprehensive imaging analysis of control and Δ123/Δe161 tumors can be found in figure 6. (II-KK) BrdU staining and quantification of the px330-control and double enhancer deletion tumors. * p=0.0003. Staining in A-W, II-JJ is H&E staining and immunohistochemistry. Staining in X-CC is in situ hybridization. Scale bars are 100um.

Supplementary Figure 9 Design of e123/e161 guide RNAs and validation of enhancer editing in mouse glioma.

(A) Schematic of NFIA enhancer loci with Sox9 and Brn2 transcription binding sites indicated in red. (B-H) Targeted Illumina Deep Sequencing of genomic DNA from Δ123/Δ161 PB-Ras, Δ123 PB-Ras, and Δ161 PB-Ras tumors harvested at P14. Graphs in B-C, F presents the number of reads that do not contain mutations (i.e. WT) and those that contain deletions or indels in e123 and/or e161 enhancers as a percentage of the total reads per sample. These data are derived from three different tumor samples that were independently processed and subjected to Deep Sequencing analysis (see methods). (D-E, G-H). Most frequent edits in the e123 and e161 regions in the Δ123/Δ161 PB-Ras, Δ123 PB-Ras, and Δ161 PB-Ras tumors detected via Deep Sequencing. Top sequence is wild-type loci, with subsequent sequences demonstrating deletion of Sox9 and Brn2 transcription binding regions at e123 and e161, respectively. Blue text denotes the sgRNA sequence and yellow text denotes the PAM sequence. Percentages represent the frequency of a given mutation/variant for a given enhancer amongst the reads presented in graphs (B-C, F) that demonstrated a deletion or indel. (I) PCR detection of enhancer deletions in PB-RAS tumors, presence of the lower bands in lanes 4 and 5 (red arrow) indicate an editing event occurred in the region of interest, and were later confirmed by next generation sequencing. Yellow arrow denotes full length enhancer fragment. Lane 1= Ladder, Lane 2= Ras Tumor, no editing guide RNAs amplified with primers 1+2, Lane 3= Ras Tumor, no editing guide RNAs amplified with primers 3+4, Lane 4= Ras tumor, edited with both sets of enhancer guide RNAs amplified with primers 1+2, Lane 5= Ras tumor, edited with both sets of enhancer guide RNAs, amplified with primers 3+4. See methods for PCR sequences. (J) Chromatin conformation capture (3C) assay performed on PB-Ras generated glioma tumors that have been subjected to CRISPR-mediated gene editing of the response regions within either e123 or e161 (individual enhancer deletion controls). Red text denotes e123 as the anchor point from which long-range DNA interactions were measured. Three independent libraries were assayed per experiment.

Supplementary Figure 10 Uncropped gel and blot Images

(A) Corresponding uncropped blots of western blots shown in Figure 2D and Supplemental Figure 4K. Uncropped blots are the size of the membrane which is shown. Membranes were often cut, as represented by dashed line, to enable blotting for multiple antibodies. The protein standards are depicted to the right of the blots. (B) Uncropped images of the DNA gels for ChIP analysis for experiments performed in Figures 3D and 3G.

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Glasgow, S., Carlson, J., Zhu, W. et al. Glia-specific enhancers and chromatin structure regulate NFIA expression and glioma tumorigenesis. Nat Neurosci 20, 1520–1528 (2017). https://doi.org/10.1038/nn.4638

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