Table 2:

Methods for Investigating Histone Modifications

Approach	Utility	Caveats	Examples from Inner Ear or Related Tissues
LC-MS	Can provide information about all of the modifications present on all of the histone proteins that are present in a sample. Can be used to compare global levels of histone modifications across experimental groups.	1. Does not link individual histones and their modifications to specific locations in the genome. 2. Additional experiments are necessary to directly link changes in modifications to any phenotype. 3. Can be costly, require special equipment, and require additional expertise in mass spectrometry.	N/A
Immunofluorescence, Western blotting	Economical, fast, and easily implemented in most laboratories. Can provide initial information and allow for correlation of various cellular processes with certain modifications that are present at high levels.	1. Variability can result from differences in fixation, section thickness, age of sample, age of antibody, antibody promiscuity, and a number of other factors making direct quantification difficult and perhaps less reliable than other methods. 2. Requires finding and validating reliable antibodies.	Mantela et al., 2005 Hansen et al., 2008 Weber et al., 2008 Chen et al. 2009 Sulg et al., 2010 Okano et al., 2011 Liu et al., 2012 Pan et al., 2013 Watanabe & Bloch 2013 Yu et al., 2013 Yue et al., 2013 Cox et al., 2014 Laos et al., 2014 Raft et al., 2014 Slattery et al., 2014 Walters et al., 2014 Huh et al., 2015 Urness et al., 2015 Chen et al., 2016 Li et al., 2017
ChlP-PCR or ChIP qPCR	Cost effective method that allows for the determination of individual histone modification at small numbers of genomic sites of interest. If the PCR is quantitative, comparisons can be made across experimental groups.	1. Is limited to the investigation of one, or at most a few, histone modifications of interest. 2. Is dependent on finding a reliable antibody against the modification of interest. 3. Only suitable for genes of interest as it does not provide genome-level information.	Abdolazimi et al., 2016 Stojanova et al., 2016 Song et al. 2017
ChlP-seq	Allows for the identification of all genomic regions that are bound to histones that have the modification of interest. Can be used to compare global levels of histone modifications across experimental groups.	1. Is dependent on finding a reliable antibody with good binding kinetics. 2. Can be costly and may require additional bioinformatics and next generation sequencing expertise.	Song et al., 2017
DamlD-seq	Requires low input of DNA. Provides whole genome sequence data including all locations of the genome where the histone modifying enzyme of interest was in close proximity to the DNA. Depending on the approach used, it is possible to generate cell type-specific expression of the adenine methyl-transferase-conjugated enzyme thus limiting the need for cell sorting.	1. Requires knowledge of the histone modifying enzyme of interest. 2. Requires genetic editing approaches to fuse adenine methyltransferase to the histone modifying enzyme of interest. 3. If the histone modifying enzyme has multiple roles involving DNA, the resulting data may include sites not related to the histone modification of interest. 4. May require additional bioinformatics and next generation sequencing expertise	N/A
Genetic or pharmacological manipulation of writer and eraser enzymes	Can uncover important processes and genes that are regulated by a histone modification of interest (or by a set of histone modifications). Genetic manipulation can be designed to be cell type specific obviating the need for cell sorting.	1. Overlap of function across a variety of histone writing and erasing enzymes as well as promiscuous activity of these enzymes on other proteins may yield complex phenotypes and can make it difficult to tie phenotypes directly to the histone modification being tested. 2. Potential off-target effects of pharmacological compounds may confound results.	Drottar et al., 2006 Stojanova et al., 2009 He et al., 2013 Yu et al., 2013 He et al., 2015 Layman et al., 2015 Li et al., 2015 Wang et al., 2015 Chen et al., 2016 He et al., 2016 Stojanova et al., 2016 Yang et al., 2017

NOTE for Table 2: All of the techniques listed above with the exception of “DamiID-seq” and “Genetic or pharmacological manipulation of writer and eraser enzymes” are ideally performed with sorted or homogeneous populations of cells or isolated single cells. Cellular heterogeneity can introduce a large degree of noise into the data resulting in incorrect estimations of the frequency and locations of histone modifications in the cell types of interest.