An expansion of the non-coding genome and its regulatory potential underlies vertebrate neuronal diversity

Abstract

Proper assembly and function of the nervous system requires the generation of a uniquely diverse population of neurons expressing a cell-type-specific combination of effector genes that collectively define neuronal morphology, connectivity, and function. How countless partially overlapping but cell-type-specific patterns of gene expression are controlled at the genomic level remains poorly understood. Here we show that neuronal genes are associated with highly complex gene regulatory systems composed of independent cell-type- and cell-stage-specific regulatory elements that reside in expanded non-coding genomic domains. Mapping enhancer-promoter interactions revealed that motor neuron enhancers are broadly distributed across the large chromatin domains. This distributed regulatory architecture is not a unique property of motor neurons but is employed throughout the nervous system. The number of regulatory elements increased dramatically during the transition from invertebrates to vertebrates, suggesting that acquisition of new enhancers might be a fundamental process underlying the evolutionary increase in cellular complexity.

Keywords: Isl1; Lhx3; cell fate specification; cellular diversity; chromatin interactions; enhancers; gene deserts; gene regulation; neuronal development; noncoding DNA.

Figure 1.. An expansion in gene regulatory complexity associated with neuronal genes.

(A) Quantifications of the total numbers of highly conserved (PhastCons > 0.5) putative regulatory elements in the proximity of top 500 genes highly induced in neuronal (motor neuron (green), sensory neuron (cyan), cortical excitatory neuron (navy), cortical PV interneuron (magenta), adult frontal cortex, cortical plate and cerebellum (teal) and embryonic brain (light blue)), non-neuronal tissues (white) and all genes (gray) in the mouse. (B) Quantifications of the total numbers of DNase accessible putative regulatory elements in the proximity of the top 500 genes highly induced in each tissue and cell type (colors are same as (A)). (C) Quantifications of the non-coding intergenic regulatory domain size associated with the top 500 genes highly induced in each tissue and cell type (colors are same as (A). (D) Cell and stage specific peaks of DNA accessibility in the proximity of the broadly expressed neuronal gene Gap43 from four distinct primary neuronal cell types (embryonic motor neuron (green); embryonic sensory neurons (cyan); adult excitatory neurons (blue); adult inhibitory neurons (magenta), shares (black) and conserved elements (gray)). (E) Complex patterns of cell specific DNA accessibility around broadly expressed neuronal genes encompassing many aspects of neuronal identity. Each tick mark represents a cell specific peak of DNA accessibility in the proximity of a given gene. (F) Genome-wide sets of all cell specific peaks associated broadly expressed neuronal genes.

Figure 2.. Motor neuron selector transcription factors globally regulate motor neuron gene expression program.

(A) Genome browser view of ~650kb window downstream of the Isl1 locus in motor neurons. Isl1 is engaged with 4 distal regions (green arcs and tick marks) distributed across the ~650kb region including three distinct enhancers (E+222, E+613, E+622) highly enriched for Mediator (blue) and H3K27ac (magenta), occupied by the motor neuron specific selector transcription factors Isl1 and Lhx3 (black tick marks) and exhibiting DNA accessibility in vivo (green). (B) Genome wide maps of all enhancer-promoter interactions ranked by interaction distance and corresponding enrichment of enhancer associated factors at two anchors (Promoter centered at x=0(left) and enhancer(right)). P-value of interaction call, raw sequencing reads for Med1(blue), H3K27ac(magenta), in vivo DNA accessibility (ATAC-seq) in e10.5 Hb9::GFP motor neurons (green) and for Isl1/Lhx3 binding at distal enhancers (black) from left to right. (C) Top three motifs enriched in engaged motor neuron enhancers. (D) Heat map displaying gene expression levels (Log2(TPM+1)) for differentially expressed genes during motor neuron specification compared to mESC (left). Binary plot showing which genes are interacting with distal Isl/Lhx3 (IL) bound enhancers (right). Black bar demarcates a gene which interacts with a distal IL bound enhancer. (E) High-resolution examples of ~2kb windows surrounding distal enhancers (colors same as 1a) bound by Isl1/Lhx3 regulating broad aspects of motor neuron identity (Isl2 (transcriptional identity), Chat (neurotransmitter identity), Slit2 (axon guidance), Nefl/m (structural/cytoskeleton)). (F) Heat map of gene expression levels for neuronal genes targeted by Isl1/Lhx3 enhancers (left, subset of Isl1/Lhx3 target genes from Figure 2D). Heat map of a selected set of Isl/Lhx3 target genes which exhibit either broad or motor neuron specific expression (right) (G) Bar plot showing broad targeting of Isl/Lhx3 enhancers across different gene categories.

Figure 3.. Distributed gene regulatory architecture in postmitotic neurons.

(A) Plots of all enhancer-promoter interactions associated with induced postmitotic motor neuron (MN) identity genes (green), motor neuron progenitor (pMN) identity genes (blue) and mouse embryonic stem (mES) cell identity genes (orange) showing motor neuron genes are regulated by distantly distributed enhancers while pMN and mES cell genes are regulated by proximally clustered enhancers. (top) Distributions for enhancer-promoter interaction distances for cell identity genes in MN (green), pMN (blue) and mES (orange) (bottom). (B) Boxplots of interaction distances for stably expressed (S, light shade) and induced (I, in one cell type relative to the two others, dark shade) motor neuron (green), pMN (blue) and mES (orange) genes. (C) Boxplots of pairwise enhancer-enhancer distances assessing distributed enhancer organization associated with motor neuron genes (green) compared to pMN (blue) and mES (orange) genes. (D) Boxplots of intergenic domain size for motor neuron (green), pMN (blue), mES (orange) and all (gray) genes. (E) Boxplots quantifying the numbers of conserved DNA elements in the proximity of motor neuron genes (green) compared to pMN (blue), mES (orange) and all (gray) genes. (F) Individual examples of expanded interaction domains around motor neuron genes. Enhancer-promoter interactions surrounding the postmitotic motor neuron gene, Nefm (green, top), neural progenitor gene Nestin (blue, middle) and embryonic stem cell gene Nanog (orange, bottom). Genome browser tracks represent enrichment of Med1 in each cell type. Black scale bars represent total interaction span for each gene.

Figure 4.. Functional dissection of distributed enhancers in motor neurons.

(A) Individual enhancers around the Isl1 gene targeted with pairs of gRNAs to selectively delete in isolation and combination all regions of enriched transcription factor binding (black), in vivo DNA accessibility (green) as well as highly conserved non-coding sequence (blue). (B) qPCR analysis of endogenous Isl1 gene expression in motor neurons upon enhancer deletion (n >=3, Data represented as the mean where error bars represent +/− SEM). (C) Immunostaining analysis of Olig2 ((green) progenitor marker, top) and Isl1((gray) postmitotic marker, bottom) protein levels upon enhancer deletion during motor neuron differentiation with patterning signals retinoic acid and sonic hedgehog agonist. (D) Quantification of decrease in Isl1 intensity upon enhancer deletion in C (n >=3).

Figure 5.. Cell-type- and cell-stage-specific enhancer activity in vivo.

(A) Genome browser view of chromatin interactions (green arcs), DNA accessibility and transcription factor binding in a ~1Mb genomic region surrounding the Isl1 gene displaying largely non-overlapping patterns of DNA accessibility in primary motor neurons (green track) and sensory neurons (cyan track) associated with motor neuron selector factors Isl1 and Lhx3 (black). (B) High resolution examples (~1-2kb) of neuron specific DNA accessibility. MN specific enhancers E+222, E+613 and E+622 are bound by Isl1 and Lhx3 and accessible specifically in primary motor neurons (green) but inaccessible in primary sensory neurons (cyan) while enhancers E-338 and E+510 exhibits SN specific DNA accessibility void of Isl1 binding in motor neurons. (C) Enhancer E+222 shows cell type specificity in spinal cord and dorsal root ganglia (DRG) with activity only in nascent and ventro-medial located Isl1+ motor neurons (magenta). Enhancer E+622 exhibits complex patterns of activity detected in ventro-medial located Isl1+ motor neurons (magenta) and nascent dorsal progenitors (n >=3). (D) Line plots of +/− 2kb window surrounding ATAC-Seq peak calls showing dynamic reorganization of patterns of DNA accessibility around motor neuron genes in vivo between e10.5 and e13.5.

Figure 6.. Evolutionary dynamics of neuronal regulatory domains.

(A) Quantifications showing fold change in relative intergenic size for orthologue matched neuronal genes compared to non-neuronal genes across seven species (vertebrates (shades of blue) and invertebrates (shades of purple)). Dashed line represents no change between the gene sets. Gray shading represents relative decrease in neuronal domain size. (B) Quantifications of increases in accessible DNA elements per gene associated with all genes (gray), transcription factors (blue) and neuronal effector genes (green) between fly (left) and mouse (right). (C) Proportions of accessible DNA elements per gene located in genic (light blue) and intergenic (red) non-coding DNA in fly (left) and mouse (right). (D) Expansion in intergenic domains and numbers of accessible DNA elements (genic elements (light blue) and intergenic elements (red)) around neuronal effector genes, Dcc/Fra (pathfinding), Grik2/GluRa (neurotransmitter receptor) and Syt1 (synaptic transmission) between mouse and fly. (E) Heatmaps representing per base 60 way vertebrate PhastCons scores in 2kb windows surrounding cell type specific peaks of DNA accessibility around broadly expressed neuronal genes in spinal motor neurons (green), cortical excitatory neurons (blue) and random genomic regions (black). (F) Composite plots representing average per base 60 way vertebrate PhastCons scores in 2kb windows surrounding cell type specific peaks of DNA accessibility around broadly expressed neuronal genes in spinal motor neurons (green), cortical excitatory neurons (blue) and random genomic regions (black). (G) Boxplots representing average per base 60 way vertebrate PhastCons scores per 200bp region surrounding cell type specific peaks of DNA accessibility around broadly expressed neuronal genes in spinal motor neurons (green), cortical excitatory neurons (blue) and random genomic regions (black).