Ting-Ya Chang, David J. Waxman
Background: STARR-seq and other massively-parallel reporter assays are widely used to discover functional enhancers in transfected cell models, which can be confounded by plasmid vector-induced type-I interferon immune responses and lack the multicellular environment and endogenous chromatin state of complex mammalian tissues. Results: We describe HDI-STARR-seq, which combines STARR-seq plasmid library delivery to the liver, by hydrodynamic tail vein injection (HDI), with reporter RNA transcriptional initiation driven by a minimal Albumin promoter, which we show is essential for mouse liver STARR-seq enhancer activity assayed 7 days after HDI. Importantly, little or no vector-induced innate type-I interferon responses were observed. Comparisons of HDI-STARR-seq activity between male and female mouse livers and in livers from males treated with an activating ligand of the transcription factor (TF) CAR (Nr1i3) identified many condition-dependent enhancers linked to condition-specific gene expression. Further, thousands of active liver enhancers were identified using a high complexity STARR-seq library comprised of ~50,000 genomic regions released by DNase-I digestion of mouse liver nuclei. When compared to stringently inactive library sequences, the active enhancer sequences identified were highly enriched for liver open chromatin regions with activating histone marks (H3K27ac, H3K4me1, H3K4me3), were significantly closer to gene transcriptional start sites, and were significantly depleted of repressive (H3K27me3, H3K9me3) and transcribed region histone marks (H3K36me3). Conclusion: HDI-STARR-seq offers substantial improvements over current methodologies for large scale, functional profiling of enhancers, including condition-dependent enhancers, in liver tissue in vivo, and can be adapted to characterize enhancer activities in a variety of species and tissues by selecting suitable tissue- and species-specific promoter sequences.
Ting-Ya Chang, David J. Waxman. HDI-STARR-seq: Condition-specific enhancer discovery in mouse liver in vivo. 2024-12-24. https://doi.org/10.1186/s12864-024-11162-9
A focused STARR-seq plasmid library targeted 100 mouse liver DNase-I hypersensitive regions, with 100 successfully cloned genomic PCR targets and additional measurable MACS2 subpeaks. The STARR-TYC3/7 library was delivered to vehicle-treated male and female mice and TCPOBOP-treated male mice by hydrodynamic tail-vein injection; liver reporter RNA and DNA were collected 7 days after delivery.
The same global liver-DNase fragment pool was cloned into STARR-TYC4 with an SCP1 promoter, STARR-TYC5 with an ORI promoter, or STARR-TYC6 with the minimal Albumin promoter. The promoter libraries were delivered to TCPOBOP-treated male mouse liver and assayed 7 days later; the available processed GEO table reports aggregate mapped RNA/DNA library activity by sample rather than element-level activities.
A high-complexity STARR-TYC6 library of DNase-I-released mouse liver genomic fragments was delivered to mouse liver by hydrodynamic injection. Reporter activity was measured 7 days later in four untreated males, four TCPOBOP-treated males and three untreated females, with plasmid/DNA input normalization and condition-specific enhancer classifications.
Original supplemental and deposited inputs retained for this study. Download files individually or together as a ZIP; nested folders are preserved. Source reuse terms apply, and sequencing reads may be omitted.
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