DOI: 10.5061/dryad.rv15dv4kg
title: Data from: Transcriptional regulation of human <em>NMNAT2</em>: Insights from 3D genome sequencing and bioinformatics

description: Nicotinamide mononucleotide adenylyl transferases 2 (NMNAT2) is a crucial
 enzyme involved in the synthesis of nicotinamide adenine dinucleotide
 (NAD), which is vital for neuronal health. NMNAT2 mRNA levels positively
 correlate with cognitive function in older adults but decline following
 various injuries or proteinopathies. To explore the transcriptional
 regulation of NMNAT2, we employed chromosome conformation capture followed
 by high-throughput sequencing (4C-seq) to identify potential enhancer and
 silencer regions interacting with the NMNAT2 promoter. Our findings
 revealed distinct NMNAT2 promoter interactomes in undifferentiated versus
 neuron-like SH-SY5Y cells. Through bioinformatics analyses—including the
 Genomic Regions Enrichment of Annotations Tool (GREAT), MEME Suite,
 WhichTF, FIMO, and RNA sequencing—we pinpointed
 potential NMNAT2-associated genes and transcription factors.
 Using data from the Religious Orders Study and the Rush Memory and Aging
 Project (ROSMAP) single-nucleus RNA sequencing, we demonstrated that the
 expression levels of many NMNAT2-associated genes significantly correlate
 with NMNAT2 in human neurons. Employing CRISPR-Cas9 strategies, we
 confirmed the necessity of two specific genomic regions within the
 interactomes and four transcription factors for regulating NMNAT2
 transcription. Together, our study identifies distinctive genomic loci
 containing NMNAT2 regulatory elements in undifferentiated versus
 neuron-like SH-SY5Y cells, as well as NMNAT2-associated genes and putative
 transcription factors.
description: # Data from: Transcriptional regulation of human *NMNAT2*: Insights from
 3D genome sequencing and bioinformatics Dataset DOI:
 [10.5061/dryad.rv15dv4kg](10.5061/dryad.rv15dv4kg) ## Description of the
 data and file structure These supplementary data include the analysis
 results, which comprise RNA-seq data from undifferentiated and
 differentiated/neuron-like SH-SY5Y cells (Supplementary Table 1); putative
 transcription factors predicted from the *NMNAT2* promoter regions using
 MEME Suite analysis (meme-suite.org/meme/tools/meme) integrated with
 RNA-seq data from undifferentiated and differentiated/neuron-like SH-SY5Y
 cells (Supplementary Table 2); putative transcription factors predicted
 from the 4C interactomes through WhichTF analysis
 (bitbucket.org/bejerano/whichtf/src/master) integrated with RNA-seq data
 from undifferentiated and differentiated/neuron-like SH-SY5Y cells
 (Supplementary Table 3); transcription factor occurrences of HSF1, ATF4,
 ATF6, and SOX4 in Region 1 and Region 2 using FIMO analysis
 (meme-suite.org/meme/tools/fimo) (Supplementary Table 4 and 5); as well as
 a list of primers and gRNAs used in this study (Supplementary Table 6).
 ### Files and variables #### File:
 SupTable1-RNA-seq_data_for_undifferentiated_and_neuron-like_SH-SY5Y_cells.xlsx **Description:** This file contains RNA-seq data from undifferentiated and differentiated/neuron-like SH-SY5Y cells. Raw sequencing data were aligned to the human reference genome – hg19. Differential expression analysis was performed using edgeR. SHSY5Y: SH-SY5Y cells. diffSHSY5Y: differentiated /neuron-like SH-SY5Y cells. FC: fold change. FDR: false discovery rate. #### File: SupTable1-RNA-seq_data_for_undifferentiated_and_neuron-like_SH-SY5Y_cells_NoFormatting.xlsx **Description:** This file contains RNA-seq data from undifferentiated and differentiated/neuron-like SH-SY5Y cells. Raw sequencing data were aligned to the human reference genome – hg19. Differential expression analysis was performed using edgeR. SHSY5Y: SH-SY5Y cells. diffSHSY5Y: differentiated /neuron-like SH-SY5Y cells. FC: fold change. FDR: false discovery rate. #### File: SupTable2-TFs_from_the_NMNAT2_promoter.xlsx **Description:** This file contains transcription factors predicted from the *NMNAT2* promoter regions (199bp region; 616bp excluding 199bp region) using MEME Suite analysis integrated with RNA-seq data from undifferentiated and differentiated/neuron-like SH-SY5Y cells. Motif analysis was carried out using MEME version 5.5.3. Any number of repetitions was used for the distribution of motif sites in sequences. The discovered motifs were input in Tomtom version 5.5.3 using the HOCOMOCOv11_core motif database and default parameters for TF prediction. SHSY5Y: SH-SY5Y cells. diffSHSY5Y: differentiated/neuron-like SH-SY5Y cells. Chr: chromosome. Start: start position of exons. End: end position of exons. Length: gene length. geneName: gene description. SHSY5Y_vs_diffSHSY5Y_FC: fold change.  SHSY5Y_vs_diffSHSY5Y_logFC: log base 2 fold change.  SHSY5Y_vs_diffSHSY5Y_PValue: p value.  SHSY5Y_vs_diffSHSY5Y_FDR: false discovery rate. CPM: counts per million. #### File: SupTable2-TFs_from_the_NMNAT2_promoter_199bpRegion_NoFormatting.xlsx **Description:** This file contains transcription factors predicted from the *NMNAT2* promoter region (199bp region) using MEME Suite analysis integrated with RNA-seq data from undifferentiated and differentiated/neuron-like SH-SY5Y cells. Motif analysis was carried out using MEME version 5.5.3. Any number of repetitions was used for the distribution of motif sites in sequences. The discovered motifs were input in Tomtom version 5.5.3 using the HOCOMOCOv11_core motif database and default parameters for TF prediction. SHSY5Y: SH-SY5Y cells. diffSHSY5Y: differentiated/neuron-like SH-SY5Y cells. Chr: chromosome. Start: start position of exons. End: end position of exons. Length: gene length. geneName: gene description. SHSY5Y_vs_diffSHSY5Y_FC: fold change.  SHSY5Y_vs_diffSHSY5Y_logFC: log base 2 fold change.  SHSY5Y_vs_diffSHSY5Y_PValue: p value.  SHSY5Y_vs_diffSHSY5Y_FDR: false discovery rate. CPM: counts per million. #### File: SupTable2-TFs_from_the_NMNAT2_promoter_616bp-199bpRegion_NoFormatting.xlsx **Description:** This file contains transcription factors predicted from the *NMNAT2* promoter region (616bp excluding 199bp region) using MEME Suite analysis integrated with RNA-seq data from undifferentiated and differentiated/neuron-like SH-SY5Y cells. Motif analysis was carried out using MEME version 5.5.3. Any number of repetitions was used for the distribution of motif sites in sequences. The discovered motifs were input in Tomtom version 5.5.3 using the HOCOMOCOv11_core motif database and default parameters for TF prediction. SHSY5Y: SH-SY5Y cells. diffSHSY5Y: differentiated/neuron-like SH-SY5Y cells. Chr: chromosome. Start: start position of exons. End: end position of exons. Length: gene length. geneName: gene description. SHSY5Y_vs_diffSHSY5Y_FC: fold change.  SHSY5Y_vs_diffSHSY5Y_logFC: log base 2 fold change.  SHSY5Y_vs_diffSHSY5Y_PValue: p value.  SHSY5Y_vs_diffSHSY5Y_FDR: false discovery rate. CPM: counts per million. #### File: SupTable3-TFs_from_the_4C_interactomes_matched_to_RNA-seq_data.xlsx **Description:** This file contains transcription factors predicted from the 4C interactomes using WhichTF analysis integrated with RNA-seq data from undifferentiated and differentiated/neuron-like SH-SY5Y cells. WhichTF analysis was performed using the default setting in the pipeline.  TF: transcription factor. log10CP: log10 conditional p-value.  SHSY5Y: SH-SY5Y cells. diffSHSY5Y: differentiated/neuron-like SH-SY5Y cells.  CPM: counts per million.  SHSY5Y_vs_diffSHSY5Y_FC: fold change.  SHSY5Y_vs_diffSHSY5Y\_ FDR: false discovery rate. #### File: SupTable3-TFs_from_the_4C_interactomes_matched_to_RNA-seq_data_NoFormatting.xlsx **Description:** This file contains transcription factors predicted from the 4C interactomes using WhichTF analysis integrated with RNA-seq data from undifferentiated and differentiated/neuron-like SH-SY5Y cells. WhichTF analysis was performed using the default setting in the pipeline.  TF: transcription factor. log10CP: log10 conditional p-value.  SHSY5Y: SH-SY5Y cells. diffSHSY5Y: differentiated/neuron-like SH-SY5Y cells.  CPM: counts per million.  SHSY5Y_vs_diffSHSY5Y_FC: fold change.  SHSY5Y_vs_diffSHSY5Y\_ FDR: false discovery rate. #### File: SupTable4-Region1_FIMO_analysis.xlsx **Description:** This file contains transcription factor occurrences of HSF1, ATF4, ATF6, and SOX4 in Region 1 using FIMO analysis. TF occurrences of HSF1, ATF4, ATF6, and SOX4 in Region 1 were predicted using FIMO version 5.5.5 with default parameters. Motifs of HSF1, ATF4, ATF6, and SOX4 were retrieved from the JASPAR 2022 motif database (jaspar2022.genereg.net). chr1: chromosome 1. start: start position of matched sequences. stop: stop position of matched sequences. #### File: SupTable4-Region1_FIMO_analysis_ATF4MA0833.1_NoFormatting.xlsx **Description:** This file contains transcription factor occurrences of ATF4 in Region 1 using FIMO analysis. TF occurrences of ATF4 in Region 1 were predicted using FIMO version 5.5.5 with default parameters. Motif of ATF4 was retrieved from the JASPAR 2022 motif database (jaspar2022.genereg.net). chr1: chromosome 1. start: start position of matched sequences. stop: stop position of matched sequences. #### File: SupTable4-Region1_FIMO_analysis_ATF4MA0833.2_NoFormatting.xlsx **Description:** This file contains transcription factor occurrences of ATF4 in Region 1 using FIMO analysis. TF occurrences of ATF4 in Region 1 were predicted using FIMO version 5.5.5 with default parameters. Motif of ATF4 was retrieved from the JASPAR 2022 motif database (jaspar2022.genereg.net). chr1: chromosome 1. start: start position of matched sequences. stop: stop position of matched sequences. #### File: SupTable4-Region1_FIMO_analysis_ATF6MA1466.1_NoFormatting.xlsx **Description:** This file contains transcription factor occurrences of ATF6 in Region 1 using FIMO analysis. TF occurrences of ATF6 in Region 1 were predicted using FIMO version 5.5.5 with default parameters. Motif of ATF6 was retrieved from the JASPAR 2022 motif database (jaspar2022.genereg.net). chr1: chromosome 1. start: start position of matched sequences. stop: stop position of matched sequences. #### File: SupTable4-Region1_FIMO_analysis_HSF1MA0486.1_NoFormatting.xlsx **Description:** This file contains transcription factor occurrences of HSF1 in Region 1 using FIMO analysis. TF occurrences of HSF1 in Region 1 were predicted using FIMO version 5.5.5 with default parameters. Motif of HSF1 was retrieved from the JASPAR 2022 motif database (jaspar2022.genereg.net). chr1: chromosome 1. start: start position of matched sequences. stop: stop position of matched sequences. #### File: SupTable4-Region1_FIMO_analysis_HSF1MA0486.2_NoFormatting.xlsx **Description:** This file contains transcription factor occurrences of HSF1 in Region 1 using FIMO analysis. TF occurrences of HSF1 in Region 1 were predicted using FIMO version 5.5.5 with default parameters. Motif of HSF1 was retrieved from the JASPAR 2022 motif database (jaspar2022.genereg.net). chr1: chromosome 1. start: start position of matched sequences. stop: stop position of matched sequences. #### File: SupTable4-Region1_FIMO_analysis_SOX4MA0867.1_NoFormatting.xlsx **Description:** This file contains transcription factor occurrences of SOX4 in Region 1 using FIMO analysis. TF occurrences of SOX4 in Region 1 were predicted using FIMO version 5.5.5 with default parameters. Motif of SOX4 were retrieved from the JASPAR 2022 motif database (jaspar2022.genereg.net). chr1: chromosome 1. start: start position of matched sequences. stop: stop position of matched sequences. #### File: SupTable4-Region1_FIMO_analysis_SOX4MA0867.2_NoFormatting.xlsx **Description:** This file contains transcription factor occurrences of SOX4 in Region 1 using FIMO analysis. TF occurrences of SOX4 in Region 1 were predicted using FIMO version 5.5.5 with default parameters. Motif of SOX4 were retrieved from the JASPAR 2022 motif database (jaspar2022.genereg.net). chr1: chromosome 1. start: start position of matched sequences. stop: stop position of matched sequences. #### File: SupTable5-Region2_FIMO_analysis.xlsx **Description:** This file contains transcription factor occurrences of HSF1, ATF4, ATF6, and SOX4 in Region 2 using FIMO analysis. TF occurrences of HSF1, ATF4, ATF6, and SOX4 in Region 2 were predicted using FIMO version 5.5.5 with default parameters. Motifs of HSF1, ATF4, ATF6, and SOX4 were retrieved from the JASPAR 2022 motif database (jaspar2022.genereg.net). chr1: chromosome 1. start: start position of matched sequences. stop: stop position of matched sequences. #### File: SupTable5-Region2_FIMO_analysis_ATF4MA0833.1_NoFormatting.xlsx **Description:** This file contains transcription factor occurrences of ATF4 in Region 2 using FIMO analysis. TF occurrences of ATF4 in Region 2 were predicted using FIMO version 5.5.5 with default parameters. Motif of ATF4 was retrieved from the JASPAR 2022 motif database (jaspar2022.genereg.net). chr1: chromosome 1. start: start position of matched sequences. stop: stop position of matched sequences. #### File: SupTable5-Region2_FIMO_analysis_ATF4MA0833.2_NoFormatting.xlsx **Description:** This file contains transcription factor occurrences of ATF4 in Region 2 using FIMO analysis. TF occurrences of ATF4 in Region 2 were predicted using FIMO version 5.5.5 with default parameters. Motif of ATF4 was retrieved from the JASPAR 2022 motif database (jaspar2022.genereg.net). chr1: chromosome 1. start: start position of matched sequences. stop: stop position of matched sequences. #### File: SupTable5-Region2_FIMO_analysis_ATF6MA1466.1_NoFormatting.xlsx **Description:** This file contains transcription factor occurrences of ATF6 in Region 2 using FIMO analysis. TF occurrences of ATF6 in Region 2 were predicted using FIMO version 5.5.5 with default parameters. Motif of ATF6 was retrieved from the JASPAR 2022 motif database (jaspar2022.genereg.net). chr1: chromosome 1. start: start position of matched sequences. stop: stop position of matched sequences. #### File: SupTable5-Region2_FIMO_analysis_HSF1MA0486.1_NoFormatting.xlsx **Description:** This file contains transcription factor occurrences of HSF1 in Region 2 using FIMO analysis. TF occurrences of HSF1 in Region 2 were predicted using FIMO version 5.5.5 with default parameters. Motif of HSF1 was retrieved from the JASPAR 2022 motif database (jaspar2022.genereg.net). chr1: chromosome 1. start: start position of matched sequences. stop: stop position of matched sequences. #### File: SupTable5-Region2_FIMO_analysis_HSF1MA0486.2_NoFormatting.xlsx **Description:** This file contains transcription factor occurrences of HSF1 in Region 2 using FIMO analysis. TF occurrences of HSF1 in Region 2 were predicted using FIMO version 5.5.5 with default parameters. Motif of HSF1 was retrieved from the JASPAR 2022 motif database (jaspar2022.genereg.net). chr1: chromosome 1. start: start position of matched sequences. stop: stop position of matched sequences. #### File: SupTable5-Region2_FIMO_analysis_SOX4MA0867.1_NoFormatting.xlsx **Description:** This file contains transcription factor occurrences of SOX4 in Region 2 using FIMO analysis. TF occurrences of SOX4 in Region 2 were predicted using FIMO version 5.5.5 with default parameters. Motif of SOX4 were retrieved from the JASPAR 2022 motif database (jaspar2022.genereg.net). chr1: chromosome 1. start: start position of matched sequences. stop: stop position of matched sequences. #### File: SupTable5-Region2_FIMO_analysis_SOX4MA0867.2_NoFormatting.xlsx **Description:** This file contains transcription factor occurrences of SOX4 in Region 2 using FIMO analysis. TF occurrences of SOX4 in Region 2 were predicted using FIMO version 5.5.5 with default parameters. Motif of SOX4 were retrieved from the JASPAR 2022 motif database (jaspar2022.genereg.net). chr1: chromosome 1. start: start position of matched sequences. stop: stop position of matched sequences. #### File: SupTable6-gRNA_and_primer_sequences.xlsx **Description:** This file contains a list of primers and gRNAs used in this study. #### File: SupTable6-gRNA_sequences_NoFormatting.xlsx **Description:** This file contains a list of gRNAs used in this study. #### File: SupTable6-primer_sequences_NoFormatting.xlsx **Description:** This file contains a list of primers used in this study. #### ## Code/software Microsoft Excel version 16.95.4