Research & Projects

Research in the Ahituv Lab focuses on understanding the role of regulatory sequences in biology and disease. Through a combination of genomic technologies, human patient samples, mouse and fish genetic engineering technologies, and massively parallel reporter assays (MPRA), we are working to elucidate mechanisms whereby genetic variation within these sequences leads to changes in phenotypes. In addition, we are also using these sequences as therapeutic targets. Our lab is broadly working on the following four themes:

Gene regulatory mutations and human disease

Using various genomic assays (RNA-seq, ChIP-seq, Cut&Tag, ATAC-seq, Hi-C, single cell RNA-seq, single cell ATAC-seq and many others) our lab is characterizing genes, genetic pathways and regulatory elements involved in human disease. These include neurodevelopmental diseases such as autism spectrum disorder (ASD) and epilepsy, obesity, adolescent idiopathic scoliosis (AIS), limb malformations, inaugural hernia and many others. Some examples are shown below:

Neurodevelopmental diseases

As part of the psychENCODE consortium, we are using various genomic tools, such as RNA-seq, ChIP-seq, ATAC-seq, single-cell RNA-seq, single-cell ATAC-seq and others to identify genes, genetic pathways and gene regulatory elements that are involved in neurodevelopmental diseases such as ASD, epilepsy and others. Candidate regulatory sequences are then functionally characterized using massively parallel reporter assays (MPRAs) that can test thousands of sequences in parallel for their regulatory activity.

Functional characterization of genes and regulatory elements involved in the differentiation of stem cells into neurons (Inoue, Kreimer et. al. Cell Stem Cell 2019)

Obesity

By using various genomic techniques (RNA-seq, ChIP-seq, Cut&Tag, single-cell RNA-seq and single-cell ATAC-seq) on mouse hypothalamus, we are characterizing in a cell specific manner genes and regulatory elements that are associated with obesity. These are followed up by mouse genetic manipulations to characterize their function.

Hypothalamus from mice treated with various leptin conditions were characterized for leptin receptor specific gene expression (RNA-seq) and regulation (ChIP-seq and ATAC-seq) (Inoue et. al. Nature Metabolism 2019)

Limb malformations

Limb malformations are the second most common form of human congenital abnormalities (prevalence of 1 in every 500 births), and very few gene mutations leading to non-syndromic/isolated limb malformations have been found. To this end, we are characterizing numerous novel limb enhancers in the human genome and collecting DNA from individuals with various limb malformations to screen them for mutations in these enhancers. More info: Limb Study.

Enhancer mutations lead to human limb malformations. (a) Photograph of the right hand of an individual that has preaxial polydactyly with triphalangeal thumb. (b) Sequencing of the Sonic Hedgehog (SHH) limb enhancer shows an insertion of 13 base pairs (bp) at the bottom compared to an unaffected individual at the top. (c) Mouse transgenic enhancer assay of a wild type SHH limb enhancer showing limb posterior activity at embryonic day 11.5 (E11.5), where Shh is normally expressed. (d) Mouse transgenic enhancer assay of the SHH limb enhancer with the 13bp insertion showing strong posterior and ectopic anterior activity at E11.5 due to the insertion. Adapted from Laurell, VanderMeer et. al. Human Mutation 2012.

Gene regulatory variation and morphological differences between species

Using various genomic assays (RNA-seq, ChIP-seq, Cut&Tag, ATAC-seq, Hi-C and many others) our lab is characterizing genes, genetic pathways and regulatory elements involved in morphological differences between species. We are particularly interested in sequences that lead to human-specific traits and also bat morphology.

Bat wing development

The bat wing is one of the most striking examples of morphological variation in vertebrates, characterized by dramatically elongated fingers and retained interdigital webbing, enabling these mammals to fly. In collaboration with Dr. Nicola Illing (University of Cape Town), we are taking advantage of or unique ability to collect staged embryos from the Natal long-fingered bat, Miniopterus natalensis, along with our existing annotated genome for this species and RNA-seq and ChIP-seq datasets for developing forelimb and hindlimb autopods, to decipher the genetic changes that led to the evolution of the bat wing 55 million years ago.

Bat wing development. (a) Miniopterus natalensis alizarin red/alcian blue stained bat embryo. Photo taken by Mandy K. Mason, University of Cape Town. (b) A bat accelerated regions showing forelimb enhancer activity in a mouse embryo only for the bat sequence.

In collaboration with Jay Shendure’s lab at the University of Washington, we are using and further developing massively parallel reporter assays (MPRAs) to characterize thousands of sequences in parallel for their regulatory activity. As members of the ENCODE and psychENCODE consortiums, we are characterizing hundreds of thousands of sequences for their regulatory activity using MPRA. We are also using MPRA to get a better understanding of the regulatory code by carrying out saturation mutagenesis on previously characterized regulatory elements, synthesizing sequences to learn regulatory grammar and characterizing en masse regulatory sequences that can have an effect on morphological differences between species.

Saturation mutagenesis of gene regulatory elements

Using saturation mutagenesis coupled with MPRA on 20 regulatory elements (10 promoters and 10 enhancers), where mutations are known to lead to human disease, we characterized the regulatory effect of over 30,000 mutations (MPRA data access portal).

Saturation mutagenesis MPRA. (a) Error-prone PCR is used to generate sequence variants in a regulatory region of interest. The resulting PCR products with ~1/100 changes compared with the template region are integrated in a plasmid library containing random tag sequences in the 3′ UTR of a reporter gene. Associations between tags and sequence variants are learned through high-throughput sequencing. High complexity MPRA libraries (50k–2M) are transfected as plasmids into cell lines of interest. RNA and DNA is collected and sequence tags are used as a readout. (b) Saturation mutagenesis MPRA of the cancer-associated Telomerase Reverse Transcriptase (TERT) promoter. Adapted from Kircher et. al. Nature Communications 2019.

Regulatory element synthesis to learn grammatical rules

We are synthesizing sequences to learn transcription factor binding site ‘grammar’ and how it affects gene regulatory element activation or repression.

Synthetic enhancer sequence design and controls. (a) Synthetic regulatory ele­ment sequences (SRESs) consist of patterns of 12 consensus binding sequences arranged homotypically (class I) or heterotypically (class II and class III) on 1 of 2 neutral, 168-bp templates. (b) Schematic of MPRA. SRESs were cloned upstream of a minimal promoter in a tagged luciferase library and then assayed in vivo using hydrodynamic tail vein injection. Livers were dissected 24 h after injection, mRNA was generated, and tags were reverse transcribed and sequenced. (c) Bimodal distribution of expression values for 4,966 SRESs. Expression values were calculated using the equation shown. Image adapted from Smith et. al. Nature Genetics 2013.

Cis regulation therapy

Over a thousand diseases are caused by mutations that alter gene-expression levels. Our lab is utilizing cis regulation therapy (CRT) to precisely target cis-regulatory elements in the genome and alter the expression levels of their target genes to rescue disease phenotypes. This approach takes advantage of nuclease-deficient gene-editing systems, such as zinc-fingers, TALEs or dCas9/CRISPR that are fused to proteins or effector domains, which can modulate gene-expression when targeted to a specific regulatory element (promoter, enhancer or silencer).

Haploinsufficiency

As a proof-of-principle for the feasibility of CRT, we targeted two genes that lead to severe obesity in humans and mice (SIM1 and MC4R) when haploinsufficient, having only one functional gene copy. By targeting the normal existing gene copy and increasing its expression level, we managed to rescue the obesity phenotype in mice (Matharu et. al. Science 2019).

A loss-of-function mutation in one allele lead to reduced amounts of mRNA and protein and can cause human disease, a condition termed haploinsufficiency. By up-regulating the existing normal allele using CRISPR-mediated activation (CRISPRa), whereby a nuclease-deficient Cas9 is fused to a transcriptional activator and targeted to a gene’s regulatory element (promoter or enhancer), the haploinsufficient phenotype could be rescued. Adapted from Matharu et. al. Science 2019.