MCB 182 Lecture 8.4 - Chromatin accessibility (ATAC-seq)

Chromatin Accessibility and Epigenetic Regulation

In this section, the speaker discusses chromatin accessibility and its role in gene regulation. They explain that chromatin accessibility is not a binary on/off state but rather exists on a spectrum between completely closed and completely open.

Definition of Chromatin Accessibility

• Chromatin accessibility is defined as the percentage of time that a given piece of the genome is being occupied by nucleosomes.

• It plays a critical role in gene regulation because it can change as a result of how cells respond to their environment.

Spectrum of Chromatin Accessibility

• Chromatin accessibility exists on a spectrum between completely closed and completely open.

• In the permissive chromatin state, chromatin can be opened just enough for transcription factors to bind partially occupied regions and recruit chromatin remodelers to displace or enhance nucleosome binding.

Chromatin Accessibility Assays

• There are several assays used to measure chromatin accessibility, including ATAC-seq, DNase-seq, and FAIRE-seq.

• ATAC-seq (Assay for Transposable Accessible Chromatin using sequencing) uses a hyperactive transposase called Tn5 to randomly cleave accessible regions of chromatin and insert sequencing adapters into those regions. It is more widely adopted than DNase-seq due to its robustness, reproducibility, ease of use, and ability to work with small input numbers.

• DNase-seq (DNase I hypersensitive site sequencing) uses an endonuclease called DNase1 to cleave DNA near pyrimidines in accessible chromatin. It requires larger input numbers than ATAC-seq and takes longer to perform.

Chromatin Accessibility Assays

This section discusses different chromatin accessibility assays, including DNase-seq, ATAC-seq, and MNase-seq.

DNase-seq

• DNase-seq was one of the first major chromatin accessibility assays used.

• The ENCODE consortium generated a lot of data using DNase-seq.

• However, it has recently fallen out of favor due to its challenging nature in practice.

ATAC-seq

• ATAC-seq is a more popular assay than DNase-seq because it is easier to work with.

• It cuts open chromatin regions and sequences nucleosome-free DNA.

MNase-seq

• MNase-seq uses micrococcal nuclease (MNase), which cleaves and eliminates accessible DNA.

• Unlike ATAC and DNase, MNase only sequences nucleosome-bound DNA.

• Initially, MNase cuts only open regions leaving nucleosomes with long genomic tails. Eventually, the enzyme clips away at the free tails of the nucleosome-bound genomic DNA such that only co-bound genomic DNA gets sequenced.

Practical Differences Between Assays

• DNase and ATAC both profile open chromatin regions.

• ATAC has higher resolution than DNase because it captures reads that map to regions between nucleosome-bound DNA.

• MNase degrades open chromatin and sequences only nucleosome-bound DNA.

Chromatin Accessibility

In this section, the speaker discusses how chromatin accessibility can be remodeled dynamically and the different approaches to achieve it.

Passive Competition of Transcription Factors

• Nucleosomes have a natural amount of turnover, and chromatin remodelers move nucleosomes around.

• During this process, transcription factors may bind to a particular region that happens to be free of a nucleosome and initiate transcription.

Passive Competition of Transcription Factors with Architectural Proteins

• Certain subsets of proteins that are not nucleosomes tend to get associated with chromatin in a transient way.

• When these proteins get released from the chromatin, other transcription factors can come in and recruit other secondary factors or sf to initiate transcription.

Binding to Enhancers

• Transcription factors bind to enhancers that are constitutionally open.

• Some additional transcription factor may be able to come in, bind to this enhancer, therefore go and recruit additional chromatin remodeling factors and further open up the region for transcription.

Pioneer Transcription Factors

• Pioneer transcription factors are able to bind to regions that have nucleosomes in them.

• They actively recruit chromatin remodelers in order to open up those regions of the chromatin for secondary factors and other non-pioneer transcription factors.

Chromatin Accessibility is Highly Dynamic

• Chromatin accessibility is highly dynamic property of cells.

• Even for constitutively expressed genes there's a lot of variation in terms of the accessibility between rows.

Overall, this section provides an overview on how chromatin accessibility can be remodeled dynamically through various approaches. The speaker explains each approach clearly using examples.

Hypothetical Single Cell Attacks Experiments

The speaker discusses hypothetical single cell attacks experiments where each individual row represents a different hypothetical cell in a population.

Nucleosome Positioning Upstream of Transcription Start Site

• Comparing the different rows of the single cell in the toxic experiment, you can see that the region upstream of the transcription start site generally has moderately well-positioned nucleosomes.

• Nucleosomes aren't completely fixed in locations so they still move around and they're relatively sparse.

• Open regions correspond to enhancers or different promoter elements.

• There still can be nucleosomes occupying positions that don't have important regulatory elements that were partially responsible for driving transcription.

Nucleosome Positioning Downstream of Transcription Start Site

• The gene body which is downstream of the transcription start site has weakly positioned nucleosomes.

• If your gene is being actively transcribed then rna pull 2 is running through the gene body of this gene.

• Nucleosomes have to be pushed out of the way in order for rna pul2 to transcribe your gene.