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Zebrafish transgenesis is conventionally performed via the tol2 transposable element system as it is the most straightforward, providing quick and easy transgenesis. Despite being commonly used in the zebrafish community, tol2 mediated transgenesis has issues that cause workflow slowdowns and delays. The biggest of these issues are the random nonspecific integrations which can occur multiple times within a genome. In order to generate a stable transgenic line with a single insertion, one must perform several generations of outcrosses and subsequent screening. Thus, while tol2 transgenesis is effective at generating founder animals, a considerable amount of time and labor is required later to isolate a single insertion line. A promising alternative is the use of phiC31 integrase, which follows the same workflow yet provides a single specific integration, removing the need for subsequent outcrossing. Unfortunately, this does not appear to be as easy and revolutionary as would be expected. The efficiency of phiC31 integrase was known to be lower than tol2, yet out of six different plasmids injected numerous times using different aliquots of phiC31 integrase mRNA, no positive insertion has ever been found. Although insertion appeared evident at first, further experiments showed that this was not the case. This method may still be useful but future evaluations will need to be conducted.

Chromatin compaction by linker histone H1 family proteins is a long-standing model for transcriptional repression. However, the biophysical and conformational details of such compaction , at the kilobase- and sub-kilobase length scale relevant to the activity of transcriptional regulatory elements, remain under debate. Rather than inferring such compaction from indirect measurements of features like DNA accessibility, we sought to directly probe sub-kilobase contacts between nearby nucleosomes. We developed an improved version of radiation-induced correlated cleavage with sequencing (RICC-seq), which we term RICC-seq 2.0, and used it in parallel with Micro-C to cross-validate our measurements of chromatin structure in both diverse cell types with different levels of linker histone and different levels of chromatin compaction, as well as a CRISPRi system for pan-H1 depletion. Using this system, we find that chromatin fiber de-compaction upon H1 depletion is global across the genome, reducing the contrast in inter-nucleosome contacts between acetylated chromatin and the rest of the genome. Surprisingly, this does not dramatically change higher-order chromatin organization such as nuclear compartments. Nevertheless, we observe a broad increase in accessibility at tens of thousands of sites and an increase in expression of over a thousand genes, which are enriched in polycomb repressive complex targets. Investigating the local chromatin compaction at upregulated genes as opposed to genes that do not change transcription, we observe that upregulated genes are not specifically de-compacted. Rather, our data support a model in which linker histone globally induces local compaction of nucleosome contacts and an increase in linker lengths, and repression by PRC1/2 is particularly dependent on these local features of chromatin architecture.